Sensitive and rapid methods of using chimeric receptors to identify autoimmune disease and assess disease severity

ABSTRACT

The present invention provides methods and compositions useful in the diagnosis and management of autoimmune diseases. In particular, the present invention provides improved methods and compositions for the diagnosis and management of Graves&#39; disease. The methods of the present invention not only avoids the need for radioactivity and are much simpler, economical, and rapid than methods traditionally used for the diagnosis of Graves&#39; disease, but also improve upon the sensitivity and detection abilities of previous luciferase-based autoantibody detection assays. Such improvements are based upon the superior performance of assays comprising a chimeric TSH receptor in the presence of a glucocorticoid including, but not limited to, dexamethasone.

STATEMENT OF RELATED APPLICATIONS

This is a Continuation-In-Part of co-pending application(s)PCT/US2008/011027 with an International Filing Date of Sep. 24, 2008which is a Continuation-In-Part of U.S. patent application Ser. No.11/906,189 (now issued as U.S. Pat. No. 8,293,879) filed on Oct. 1, 2007which is a Continuation-In-Part of U.S. patent application Ser. No.10/996,961 filed on Nov. 24, 2004 (now abandoned) which is a divisionalof U.S. patent application Ser. No. 09/539,735 (now issued as U.S. Pat.No. 6,852,546) filed on Mar. 30, 2000.

FIELD OF THE INVENTION

The present invention provides methods and compositions useful in thediagnosis of autoimmune diseases. In particular, the present inventionprovides methods and compositions for use in the diagnosis andmanagement of Graves' disease. For example, one composition comprises achimeric thyroid stimulating hormone receptor having improvedsensitivity and specificity for circulating thyroid stimulatingimmunoglobulin. Assays using such chimeric receptors can be optimized inthe presence of a glucocorticoid.

BACKGROUND OF THE INVENTION

Graves' disease (also referred to as “diffuse toxic goiter”), is theleading cause of hyperthyroidism due to the action of autoantibodiesthat recognize and bind to receptors present on the thyroid gland,resulting in gland growth and over-production of thyroid hormone.Graves' disease is reported to be the most frequent cause ofhyperthyroidism in childhood and adolescence (See, Boter and Brown, J.Pediatr. 132:612-618 (1998)).

Current diagnostic techniques for Graves' disease leave much to bedesired. In general, the commercially available methods are cumbersomeand laborious. Other methods require the administration of radioactivetracers to the person requiring a diagnosis. Most importantly, however,the vast majority of the presently used methods lack sufficientsensitivity such that a quick, accurate and cost-effective test can beperformed.

What is still needed is an assay system for Graves' disease that issafe, easy to use, sensitive, specific, and cost-effective.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions useful in thediagnosis and management of autoimmune diseases. In particular, thepresent invention provides methods and compositions for the diagnosisand management of Graves' disease. For example, one compositioncomprises a chimeric thyroid stimulating hormone receptor havingimproved sensitivity and specificity for circulating thyroid stimulatingimmunoglobulin. Assays using such chimeric receptors can be optimized inthe presence of a glucocorticoid.

In one embodiment, the present invention contemplates a method,comprising: a) providing; i) a cell line comprising a stably transfectedvector encoding a chimeric TSH receptor and a luciferase gene; ii) aserum sample derived from a patient suspected of having Graves' disease;and iii) a cell culture medium comprising a glucocorticoid; b)contacting the serum sample with the cell line and the medium underconditions such that the luciferase gene emits a detectable signal. Inone embodiment, the method further comprises step c) measuring thesignal intensity, wherein the intensity correlates with a thyrotropinstimulating hormone receptor autoantibody concentration present in thesample. In one embodiment, the glucocorticoid is selected from the groupcomprising dexamethasone, prednisone, hydrocortisone, fluticasone, orcortisone. In one embodiment, the contacting further comprisespolyethylene glycol. In one embodiment, the chimeric TSH receptorcomprises an amino acid sequence derived from rat chorionic hormonegonadotropin receptor. In one embodiment, the amino acid sequencecomprises seventy three amino acids corresponding to amino acid residues262-335 of a human TSH receptor amino acid sequence. In one embodiment,the serum sample comprises TSH receptor autoantibodies. In oneembodiment, the autoantibodies comprise TSH stimulating autoantibodies.In one embodiment, the autoantibodies comprise TSH blocking antibodies.

In one embodiment, the present invention contemplates a kit comprising achimeric TSH receptor and a luciferin-luciferase system capable ofdetecting serum TSH autoantibodies, wherein the system comprises aglucocorticoid. In one embodiment, the glucocorticoid is selected fromthe group comprising dexamethasone, prednisone, hydrocortisone,fluticasone, or cortisone. In one embodiment, the receptor comprises ahuman TSH receptor amino acid sequence. In one embodiment, the receptorcomprises a rat chorionic hormone receptor amino acid sequence. In oneembodiment, the rat receptor amino acid sequence comprises amino acidresidues 262-335. In one embodiment, the kit further comprises a cellline capable of expressing the chimeric TSH receptor and theluciferin-luciferase system. In one embodiment, the kit furthercomprises polyethylene glycol. In one embodiment, the kit comprises avector encoding the chimeric TSH receptor and a luciferase gene. In oneembodiment, the vector further comprises a promoter in operably linkedto the vector. In one embodiment, the promoter comprises a glycoproteinalpha subunit promoter. In one embodiment, the cell line comprises CHOcells. In one embodiment, the cell line comprises RD cells. In oneembodiment, the kit further comprises an instruction sheet.

In one embodiment, the present invention provides methods fordetermining the presence of thyroid-stimulating autoantibodies in a testsample, comprising: a) providing i) a test sample suspected ofcontaining thyroid-stimulating autoantibodies, ii) cultured cellscomprising a glucocorticoid contained within a testing means, whereinthe cells express a chimeric TSH receptor and a luciferin-luciferasesystem, and iii) polyethylene glycol; b) exposing the test sample to thecultured cells and polyethylene glycol under conditions such thatthyroid-stimulating antibodies are detectable using aluciferin-luciferase system; and c) observing for the presence ofdetectable thyroid-stimulating antibodies. In one embodiment, theglucocorticoid is selected from the group comprising dexamethasone,prednisone, hydrocortisone, fluticasone, or cortisone. In one preferredembodiment, the cultured cells are selected from the group consisting ofRDluc and CHORluc cells. In another embodiment, the observing isconducted using a luminometer. In further embodiments, the cAMPconcentration is determined by the luciferin-luciferase system. In yetanother embodiment, the methods further comprise a Growth Medium, whilein other embodiments, the methods further comprise a Stimulation Medium.In some particularly preferred embodiments, the cultured cells areexposed to the Growth Medium prior to exposure of the test sample. Instill further embodiments, the cultured cells are exposed to StimulationMedium containing the test sample. In other particularly preferredembodiments, the Stimulation Medium comprises polyethylene glycol.

The present invention also provides methods for determining the presenceof thyroid-stimulating autoantibodies in a test sample, comprising: a)providing; i) a test sample suspected of containing thyroid-stimulatingautoantibodies, ii) cultured cells comprising a glucocorticoid, whereinthe cells are selected from the group comprising RD-Rluc or CHO-Rluccells contained within a testing means, wherein the cells express achimeric TSH receptor, and iii) polyethylene glycol; b) exposing thetest sample to the cultured cells and the polyethylene glycol underconditions such that thyroid stimulating antibodies are detectable usinga luciferin-luciferase system; and c) observing for the presence ofdetectable thyroid-stimulating antibodies, wherein observing isconducted using a luminometer. In one embodiment, the glucocorticoid isselected from the group comprising dexamethasone, prednisone,hydrocortisone, fluticasone, or cortisone. In further embodiments, thecAMP concentration is determined by the luciferin-luciferase system. Insome embodiments, the methods further comprise a Growth Medium, while inother embodiments the methods further comprise a Stimulation Medium. Insome particularly preferred embodiments, the cultured cells are exposedto the Growth Medium prior to exposure of the test sample. In stillother embodiments, the cultured cells are exposed to the StimulationMedium containing the test sample. In yet other preferred embodiments,the Stimulation Medium comprises polyethylene glycol.

The present invention also provides methods for determining the presenceof thyroid-stimulating autoantibodies in a test sample, comprising: a)providing; i) a test sample suspected of containing thyroid-stimulatingautoantibodies, ii) cultured cells comprising a glucocorticoid, whereinthe cells are selected from the group comprising RD-Rluc or CHO-Rluccells contained within a testing means, wherein the cells express achimeric TSH receptor, iii) Growth Medium, and iv) Stimulation Medium,wherein the Stimulation Medium comprises polyethylene glycol; b)exposing the cultured cells to Growth Medium to produce grown cells; c)exposing the test sample to the grown cells and Stimulation Medium underconditions such that thyroid-stimulating antibodies are detectable usingthe luciferin-luciferase system; and d) observing for the presence ofdetectable thyroid-stimulating antibodies, wherein said observing isconducted using a luminometer. In one embodiment, the glucocorticoid isselected from the group comprising dexamethasone, prednisone,hydrocortisone, fluticasone, or cortisone. In further embodiments, thecAMP concentration is determined by the luciferase-luciferin system.

In one embodiment, the present invention contemplates utilizing aclinical activity score (CAS) together with the various detectionmethods described herein. CAS is a validated scoring system, designed todistinguish inflammatory from noninflammatory Graves' orbitopathy (GO),and has a high predictive value for the outcome of immunosuppressivetreatment in GO patients. It is based on the classical signs ofinflammation: pain (2 points), redness (2 points), swelling (4 points)and impaired function (2 points). While the scoring can be done based ona single examination, it is preferred that two consecutive clinicalexaminations be done and then an “activity score” can be determined,ranging from 0 to 10 points. In some embodiments wherein CAS isdetermined in a single session, a modified CAS system is employedcomprising seven items (the “7 item CAS”). The two methods differ inevaluation of visual acuity, diplopia and proptosis, which are part ofthe full 10 item CAS and are not included in the 7 item CAS. In studiesperformed before the introduction of the CAS scoring system, theophthalmopathy index (OI) as proposed by Donaldson et al or the totaleye score (TES) were developed. Secondary outcomes included the NOSPECSscheme (mnemonic for No signs or symptoms, Only signs, Soft tissueinvolvement, Proptosis, Extraocular muscle involvement, Cornealinvolvement and Sight loss, graded as O, A, B or C), diplopia,proptosis, optic neuropathy in either eye, subjective outcome measures(e.g. cosmetic response satisfaction), visual acuity, and local eyeirritation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides results for serial 3-fold dilutions of three Graves'disease IgG samples (from untreated Graves' patients), in assaysutilizing Stimulation Medium containing 6% PEG-8000.

FIG. 2 provides a comparison of CHO-Rluc luciferase results with theFRTL-5 cAMP results for IgGs from 35 untreated Graves' patients.

FIG. 3 provides a comparison of CHO-Rluc luciferase results with CHO-RcAMP results for IgGs from 35 untreated Graves' patients.

FIG. 4 provides a comparison of CHO-R cAMP results with FRTL-5 cAMPresults for IgGs from 35 untreated Graves' patients.

FIG. 5 shows the linearity of the response to bTSH of the CHO-Rluccells.

FIG. 6 shows the results for a group of samples with known TSI resultsusing FRTL-5 cells (10 μl samples of LCA TSI specimens).

FIG. 7 shows the results for a group of normal samples (10 μl of AML“normal” specimens).

FIG. 8 shows one embodiment of a DNA sequence for a chimeric hTSH/mLH(Mc4) receptor comprising 2,324 base pairs and encoding 730 amino acids(SEQ ID NO: 3). The underlined letters are the human TSHR sequence. Theletters in italics are the rat LHR sequence. “*” (T) in the rat LHRsequence is a G in the wild type sequence. This G to T mutation resultedin an amino acid change from Arginine to Serine.

FIG. 9 shows one embodiment of a 236 nucleotide glycoprotein alphasubunit promoter comprising a cyclic AMP (cAMP) regulatory element (CRE)(AF401991) sequence alignment (SEQ ID NO: 4) with a GPH promoteramplified by PCR from HEK cells (SEQ ID NO: 5). Shaded areas indicatehomology. Non-highlighted areas designate the flanking region of thepromoter in the plasmid.

FIG. 10 presents exemplary data showing the response of the CHO-RMc4,RD-RMc4 and CHO-RLuc cell lines to negative and positive TSI-containingsera.

FIG. 10A: Luciferase assay on CHO-RLuc and CHO-RMc4 cell lines inducedwith TSI negative and positive sera.

FIG. 10B: The ratio of S/N derived from the luciferase assay on CHO-RLucand CHO-RMc4 cell lines induced with TSI negative and positive sera.

FIG. 10C: Luciferase assay on CHO-RLuc and RD-RMc4 cell lines inducedwith TSI negative and positive sera.

FIG. 10D: The ratio of S/N derived from the luciferase assay on CHO-RLucand RD-RMc4 cell lines induced with TSI negative and positive sera.

FIG. 10E: The ratio of S/N derived from the luciferase assay onCHO-RLuc, CHO-RMc4 and RD-RMc4 cell lines induced with TSI negative andpositive sera.

FIG. 11 presents exemplary data showing signal-to-noise (S/N) ratios forRD-RMc4 and CHO-RLuc cell lines in response to a serum dilution profile.

FIG. 11A: The S/N ratio from the luciferase assay on CHO-RLuc andRD-RMc4 cell lines induced with same dilutions of the TSI positiveserum.

FIG. 11B: The S/N ratio from the luciferase assay on CHO-RMc4 cell lineinduced with dilution of the TSI positive serum.

FIG. 11C: The S/N ratio from the luciferase assay on CHO-RMc4 cell lineinduced with higher dilutions of the TSI positive

FIG. 12 presents exemplary data comparing TSH sensitivity between aCHO-RMc4 cell line and a CHO-RLuc cell line.

FIG. 13 presents exemplary data presenting the distribution ofsignal-to-noise ratios from human sera using CHO-RMc4 and CHO-RLuc celllines.

FIG. 14 presents exemplary data showing the relative sensitivity of theCHO-RMc4, RD-RMc4 and CHO-RLuc cell lines to clinical patient serumsamples.

FIG. 15 presents exemplary amino acid sequences: luteinizing hormonereceptor:

FIG. 15A: Callithrix jacchus (white-tufted-ear marmoset) CAJ57370 (SEQID NO: 6)

FIG. 15B: Coturnix japonica (Japanese quail) AAB32614 (SEQ ID NO: 7)

FIG. 15C: Gallus gallus (chicken) NP_(—)990267 (SEQ ID NO: 8)

FIG. 15D: Mus musculus (mouse) AAB24402 (SEQ ID NO: 9)

FIG. 15E: Bos taurus (cow) NP_(—)776806 (SEQ ID NO: 10)

FIG. 16 illustrates a representative arrangement of TSI samples in atesting plate.

FIG. 17 present exemplary Relative Light Unit (RLU) data showing thatthe alternative glucocorticoids fluticasone, prednisone, hydrocortisoneand cortisone provide equal signal intensities of the CHO-RMc4 assaywhen compared to 40 μM dexamethasone.

FIG. 18 present exemplary Serum Reference Unit percentages (SSR %) datashowing that the alternative glucocorticoids fluticasone, prednisone,hydrocortisone and cortisone provide an improved CHO-RMc4 assay.

DEFINITIONS

The terms “sample” and “specimen” in the present specification andclaims are used in their broadest sense. On the one hand, they are meantto include a specimen or culture. On the other hand, they are meant toinclude both biological and environmental samples. These terms encompassall types of samples obtained from humans and other animals, includingbut not limited to, body fluids (e.g., blood), as well as solid tissue.

Biological samples may be animal, including human, fluid or tissue, foodproducts and ingredients such as dairy items, vegetables, meat and meatby-products, and waste. These examples are not to be construed aslimiting the sample types applicable to the present invention.

As used herein, the term “kit” is used in reference to a combination ofreagents and other materials.

As used herein, the term “antibody” is used in reference to anyimmunoglobulin molecule that reacts with a specific antigen. It isintended that the term encompass any immunoglobulin (e.g., IgG, IgM,IgA, IgE, IgD, etc.) obtained from any source (e.g., humans, rodents,non-human primates, caprines, bovines, equines, ovines, etc.).

As used herein, the term “antigen” is used in reference to any substancethat is capable of reacting with an antibody. It is intended that thisterm encompass any antigen and “immunogen” (i.e., a substance whichinduces the formation of antibodies). Thus, in an immunogenic reaction,antibodies are produced in response to the presence of an antigen(immunogen) or portion of an antigen.

As used herein, the terms “antigen fragment” and “portion of an antigen”are used in reference to a portion of an antigen. Antigen fragments orportions may occur in various sizes, ranging from a small percentage ofthe entire antigen to a large percentage, but not 100% of the antigen.However, in situations where at least a portion of an antigen isspecified, it is contemplated that the entire antigen may be present. Itis contemplated that antigen fragments or portions, may, but are notrequired to comprise an “epitope” recognized by an antibody. Antigenfragments or portions also may or may not be immunogenic.

As used herein, the term “autoantibodies” refers to antibodies that arecapable of reacting against an antigenic constituent of an individual'sown tissue or cells (e.g., the antibodies recognize and bind to “self”antigens).

As used herein, the term “immunoassay” is used in reference to anymethod in which antibodies are used in the detection of an antigen. Itis contemplated that a range of immunoassay formats be encompassed bythis definition, including but not limited to, direct immunoassays,indirect immunoassays, and “sandwich” immunoassays.” However, it is notintended that the present invention be limited to any particular format.It is contemplated that other formats, including radioimmunoassays(RIA), immunofluorescent assays (IFA), and other assay formats,including, but not limited to, variations on the ELISA, RIA and/or IFAmethods will be useful in the method of the present invention.

As used herein, the term “capture antibody” refers to an antibody thatis used to bind an antigen and thereby permit the recognition of theantigen by a subsequently applied antibody. For example, the captureantibody may be bound to a microtiter well and serve to bind an antigenof interest present in a sample added to the well. Another antibody(termed the “primary antibody”) is then used to bind to theantigen-antibody complex, in effect to form a “sandwich” comprised ofantibody-antigen-antibody complex. Detection of this complex can beperformed by several methods. The primary antibody may be prepared witha label such as biotin, an enzyme, a fluorescent marker, orradioactivity, and may be detected directly using this label.Alternatively, a labelled “secondary antibody” or “reporter antibody”which recognizes the primary antibody may be added, forming a complexcomprised of an antibody-antigen-antibody-antibody complex. Again,appropriate reporter reagents are then added to detect the labelledantibody. Any number of additional antibodies may be added as desired.These antibodies may also be labelled with a marker, including, but notlimited to an enzyme, fluorescent marker, or radioactivity.

As used herein, the term “reporter reagent” or “reporter molecule” isused in reference to compounds which are capable of detecting thepresence of antibody bound to antigen. For example, a reporter reagentmay be a calorimetric substance attached to an enzymatic substrate. Uponbinding of antibody and antigen, the enzyme acts on its substrate andcauses the production of a color. Other reporter reagents include, butare not limited to, fluorogenic and radioactive compounds or molecules.This definition also encompasses the use of biotin and avidin-basedcompounds (e.g., including, but not limited to, neutravidin andstreptavidin) as part of the detection system. In one embodiment of thepresent invention, biotinylated antibodies may be used in the presentinvention in conjunction with avidin-coated solid support.

As used herein the term “signal” is used in reference to an indicatorthat a reaction has occurred, for example, binding of antibody toantigen. It is contemplated that signals in the form of radioactivity,fluorogenic reactions, luminescent and enzymatic reactions will be usedwith the present invention. The signal may be assessed quantitatively aswell as qualitatively.

As used herein the term “signal intensity” refers to magnitude of thesignal strength wherein the intensity correlates with the amount ofreaction substrate. For example, a luciferin-luciferase system generatesa signal intensity that correlates with the amount of cAMP generated bythyrotropin stimulating hormone receptor autoantibodies.

The term “correlates” indicates that a phenomenon (e.g. signalintensity) is related to another phenomenon (e.g antibody concentration,or disease severity). The relationship is typically a parallelrelationship (e.g. as one increases, the other increases).

As used herein, the term “clinical activity” means ongoing signs andsymptoms of inflammation and pathology (pain, red eyes, double vision,etc.).

As used herein, the term “luciferin-luciferase system” refers to anyprocess or method that allows the contact of luciferin and luciferase inthe presence of a substrate (i.e., for example, cAMP) under conditionssuch that the resulting luminesence may be detected. Such a system maybe comprised within a transfected host cell encoded by a vector, orprovided in separate kit containers whereby the contents may be mixedtogether.

As used herein, the term “solid support” is used in reference to anysolid material to which reagents such as antibodies, antigens, and othercompounds may be attached. For example, in the ELISA method, the wellsof microtiter plates often provide solid supports. Other examples ofsolid supports include microscope slides, coverslips, beads, particles,cell culture flasks, as well as many other items.

As used herein, the term “cell staining” is used in reference to methodsused to label or stain cells to enhance their visualization. Thisstaining or labelling may be achieved through the use of variouscompounds, including but not limited to, fluorochromes, enzymes, gold,and iodine. It is contemplated that the definition encompasses suchmethods as “in situ chromogenic assays,” in which a test (i.e., anassay) is conducted on a sample in situ. It is also contemplated thatthe in situ chromogenic assay will involve the use of an immunoassay(i.e., an ELISA).

As used herein, the term “Growth Medium” refers to a culture mediumformulated to contain various growth factors including, but not limitedto, vitamins, amino acids, co-factors, and any other appropriatenutrients to enhance growth and replication of cells in culture.

As used herein, the term “Stimulation Medium” refers to a mediumformulated to be deficient in certain constituents (e.g., sodiumchloride), in order to enhance the stimulation of by TSH and/or TSI,thereby increasing the resulting signal (e.g., cAMP and/or luciferase).

As used herein, the term “Starvation Medium” refers to a mediumformulated to be deficient in at least one growth factors included inthe Growth Medium. In preferred embodiments, this medium contains onlythe salts and glucose necessary to sustain cells for a short period oftime.

As used herein, the term “organism” and “microorganism,” are used torefer to any species or type of microorganism, including but not limitedto viruses and bacteria, including rickettsia and chlamydia. Thus, theterm encompasses, but is not limited to DNA and RNA viruses, as well asorganisms within the orders Rickettsiales and Chlamydiales.

As used herein, the term “culture,” refers to any sample or specimenwhich is suspected of containing one or more microorganisms. “Purecultures” are cultures in which the organisms present are only of onestrain of a particular genus and species. This is in contrast to “mixedcultures,” which are cultures in which more than one genus and/orspecies of microorganism are present.

As used herein, the term “cell type,” refers to any cell, regardless ofits source or characteristics.

As used herein, the term “cell line,” refers to cells that are culturedin vitro, including primary cell lines, finite cell lines, continuouscell lines, and transformed cell lines.

As used herein, the terms “primary cell culture,” and “primary culture,”refer to cell cultures that have been directly obtained from animal orinsect tissue. These cultures may be derived from adults as well asfetal tissue.

As used herein, the term “finite cell lines,” refer to cell culturesthat are capable of a limited number of population doublings prior tosenescence.

As used herein, the term “continuous cell lines,” refer to cell culturesthat have undergone a “crisis” phase during which a population of cellsin a primary or finite cell line apparently ceases to grow, but yet apopulation of cells emerges with the general characteristics of areduced cell size, higher growth rate, higher cloning efficiency,increased tumorigenicity, and a variable chromosomal complement. Thesecells often result from spontaneous transformation in vitro. These cellshave an indefinite lifespan.

As used herein, the term “transformed cell lines,” refers to cellcultures that have been transformed into continuous cell lines with thecharacteristics as described above. Transformed cell lines can bederived directly from tumor tissue and also by in vitro transformationof cells with whole virus (e.g., SV40 or EBV), or DNA fragments derivedfrom a transforming virus using vector systems.

As used herein, the term “hybridomas,” refers to cells produced byfusing two cell types together. Commonly used hybridomas include thosecreated by the fusion of antibody-secreting B cells from an immunizedanimal, with a malignant myeloma cell line capable of indefinite growthin vitro. These cells are cloned and used to prepare monoclonalantibodies.

As used herein, the term “mixed cell culture,” refers to a mixture oftwo types of cells. In some preferred embodiments, the cells are celllines that are not genetically engineered, while in other preferredembodiments the cells are genetically engineered cell lines. In someembodiments, the one or more of the cell types is “permissive” (i.e.,virus is capable of replication and spread from cell to cell within theculture). The present invention encompasses any combination of celltypes suitable for the detection, identification, and/or quantitation ofviruses in samples, including mixed cell cultures in which all of thecell types used are not genetically engineered, mixtures in which one ormore of the cell types are genetically engineered and the remaining celltypes are not genetically engineered, and mixtures in which all of thecell types are genetically engineered.

As used herein, the term “suitable for the detection of intracellularparasites,” refers to cell cultures that can be successfully used todetect the presence of an intracellular parasite in a sample. Inpreferred embodiments, the cell cultures are capable of maintainingtheir susceptibility to infection and/or support replication of theintracellular parasite. It is not intended that the present invention belimited to a particular cell type or intracellular parasite.

As used herein, the term “susceptible to infection” refers to theability of a cell to become infected with virus or another intracellularorganism. Although it encompasses “permissive” infections, it is notintended that the term be so limited, as it is intended that the termencompass circumstances in which a cell is infected, but the organismdoes not necessarily replicate and/or spread from the infected cell toother cells. The phrase “viral proliferation,” as used herein describesthe spread or passage of infectious virus from a permissive cell type toadditional cells of either a permissive or susceptible character.

As used herein, the terms “monolayer,” “monolayer culture,” and“monolayer cell culture,” refer to cells that have adhered to asubstrate and grow as a layer that is one cell in thickness. Monolayersmay be grown in various vessels including, but not limited to, flasks,tubes, coverslips (e.g., shell vials), roller bottles, etc. Cells mayalso be grown attached to microcarriers, including but not limited tobeads.

As used herein, the term “suspension,” and “suspension culture,” refersto cells that survive and proliferate without being attached to asubstrate. Suspension cultures are typically produced usinghematopoietic cells, transformed cell lines, and cells from malignanttumors.

As used herein, the terms “culture media,” and “cell culture media,”refers to media that are suitable to support the growth of cells invitro (i.e., cell cultures). It is not intended that the term be limitedto any particular culture medium. For example, it is intended that thedefinition encompass outgrowth as well as maintenance media. Indeed, itis intended that the term encompass any culture medium suitable for thegrowth of the cell cultures of interest.

As used herein, the term “obligate intracellular parasite,” (or“obligate intracellular organism) refers to any organism which requiresan intracellular environment for its survival and/or replication.Obligate intracellular parasites include viruses, as well as many otherorganisms, including certain bacteria including, but not limited to,most members of the orders: i) Rickettsiales: for example, Coxiella,Rickettsia and Ehrlichia; and ii) Chlamydiales: for example, C.trachomatis, C. psittaci. The term “intracellular parasite,” refers toany organism that may be found within the cells of a host animal,including but not limited to obligate intracellular parasites brieflydescribed above. For example, intracellular parasites include organismssuch as Brucella, Listeria, Mycobacterium (e.g., M. tuberculosis and M.leprae), and Plasmodium, as well as Rochalirnea.

As used herein, the term “antimicrobial,” is used in reference to anycompound which inhibits the growth of, or kills microorganisms. It isintended that the term be used in its broadest sense, and includes, butis not limited to compounds such as antibiotics which are producednaturally or synthetically. It is also intended that the term includescompounds and elements that are useful for inhibiting the growth of, orkilling microorgamsms.

As used herein, the terms “chromogenic compound,” and “chromogenicsubstrate,” refer to any compound useful in detection systems by theirlight absorption or emission characteristics. The term is intended toencompass any enzymatic cleavage products, soluble, as well asinsoluble, which are detectable either visually or with opticalmachinery. Included within the designation “chromogenic” are allenzymatic substrates which produce an end product which is detectable asa color change. This includes, but is not limited to any color, as usedin the traditional sense of “colors,” such as indigo, blue, red, yellow,green, orange, brown, etc., as well as fluorochromic or fluorogeniccompounds, which produce colors detectable with fluorescence (e.g., theyellow-green of fluorescein, the red of rhodamine, etc.). It is intendedthat such other indicators as dyes (e.g., pH) and luminogenic compoundsbe encompassed within this definition.

As used herein, the commonly used meaning of the terms “pH indicator,”“redox indicator,” and “oxidation-reduction indicator,” are intended.Thus, “pH indicator,” encompasses all compounds commonly used fordetection of pH changes, including, but not limited to phenol red,neutral red, bromthymol blue, bromcresol purple, bromcresol green,bromchlorophenol blue, m-cresol purple, thymol blue, bromcresol purple,xylenol blue, methyl red, methyl orange, and cresol red. The terms“redox indicator,” and “oxidation-reduction indicator,” encompasses allcompounds commonly used for detection of oxidation/reduction potentials(i.e., “eH”) including, but not limited to various types or forms oftetrazolium, resazurin, and methylene blue.

As used herein, the term “inoculating suspension,” or “inoculant,” isused in reference to a suspension which may be inoculated with organismsto be tested. It is not intended that the term “inoculating suspension,”be limited to a particular fluid or liquid substance. For example,inoculating suspensions may be comprised of water, saline, or an aqueoussolution. It is also contemplated that an inoculating suspension mayinclude a component to which water, saline or any aqueous material isadded. It is contemplated in one embodiment, that the componentcomprises at least one component useful for the intended microorganism.It is not intended that the present invention be limited to a particularcomponent.

As used herein, the term “primary isolation,” refers to the process ofculturing organisms directly from a sample. As used herein, the term“isolation,” refers to any cultivation of organisms, whether it beprimary isolation or any subsequent cultivation, including “passage,” or“transfer,” of stock cultures of organisms for maintenance and/or use.

As used herein, the term “presumptive diagnosis,” refers to apreliminary diagnosis which gives some guidance to the treatingphysician as to the etiologic organism involved in the patient'sdisease. Presumptive diagnoses are often based on “presumptiveidentifications,” which as used herein refer to the preliminaryidentification of a microorganism.

As used herein, the term “definitive diagnosis,” is used to refer to afinal diagnosis in which the etiologic agent of the patient's diseasehas been identified. The term “definitive identification” is used inreference to the final identification of an organism to the genus and/orspecies level.

The term “recombinant DNA molecule,” as used herein refers to a DNAmolecule which is comprised of segments of DNA joined together by meansof molecular biological techniques.

DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region (enhancer elements can exert their effecteven when located 3′ of the promoter element and the coding region).Transcription termination and polyadenylation signals are located 3′ ordownstream of the coding region.

The term “an oligonucleotide having a nucleotide sequence encoding agene,” refers to a DNA sequence comprising the coding region of a geneor, in other words, the DNA sequence which encodes a gene product. Thecoding region may be present in either a cDNA or genomic DNA form.Suitable control elements such as enhancers, promoters, splicejunctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the vectors ofthe present invention may contain endogenous enhancers and/or promoters,splice junctions, intervening sequences, polyadenylation signals, etc.or a combination of both endogenous and exogenous control elements.

The term “transcription unit,” as used herein refers to the segment ofDNA between the sites of initiation and termination of transcription andthe regulatory elements necessary for the efficient initiation andtermination. For example, a segment of DNA comprising anenhancer/promoter, a coding region, and a termination andpolyadenylation sequence comprises a transcription unit.

The term “regulatory element,” as used herein refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

The terms “reporter gene construct,” or “reporter gene vector,” as usedherein refers to a recombinant DNA molecule containing a sequenceencoding the product of a reporter gene and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Eukaryotic cells are known toutilize promoters, enhancers, and termination and polyadenylationsignals.

The term “reporter gene,” refers to an oligonucleotide having a sequenceencoding a gene product (typically an enzyme) which is easily andquantifiably assayed when a construct comprising the reporter genesequence operably linked to a heterologous promoter and/or enhancerelement is introduced into cells containing (or which can be made tocontain) the factors necessary for the activation of the promoter and/orenhancer elements. Examples of reporter genes include but are notlimited to bacterial genes encoding β-galactosidase (lacZ, the bacterialchloramphenicol acetyltransferase (cat) genes, firefly luciferase genesand genes encoding β-glucuronidase (GUS).

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis, et al., Science 236:1237 (1987)). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells and viruses(analogous control elements, i.e., promoters, are also found inprokaryotes). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview see Voss, et al., Trends Biochem. Sci., 11:287 (1986), andManiatis, et al., supra (1987)). For example, the SV40 early geneenhancer is very active in a wide variety of cell types from manymammalian species and has been widely used for the expression ofproteins in mammalian cells (Dijkema, et al., EMBO J. 4:761 (1985)). Twoother examples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor 1α gene(Uetsuki et al., J. Biol. Chem., 264:5791 (1989); Kim et al., Gene91:217 (1990); and Mizushima and Nagata, Nuc. Acids. Res., 18:5322(1990)) and the long terminal repeats of the Rous sarcoma virus (Gormanet al., Proc. Natl. Acad. Sci. USA 79:6777 (1982)), and the humancytomegalovirus (Boshart et al., Cell 41:521 (1985)).

The term “promoter/enhancer,” denotes a segment of DNA which containssequences capable of providing both promoter and enhancer functions (forexample, the long terminal repeats of retroviruses contain both promoterand enhancer functions). The enhancer/promoter may be “endogenous,” or“exogenous,” or “heterologous.” An endogenous enhancer/promoter is onewhich is naturally linked with a given gene in the genome. An exogenous(heterologous) enhancer/promoter is one which is placed in juxtapositionto a gene by means of genetic manipulation (i.e., molecular biologicaltechniques).

The presence of “splicing signals,” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York (1989), pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires signals directing the efficient termination and polyadenylationof the resulting transcript. Transcription termination signals aregenerally found downstream of the polyadenylation signal and are a fewhundred nucleotides in length. The term “poly A site,” or “poly Asequence,” as used herein denotes a DNA sequence which directs both thetermination and polyadenylation of the nascent RNA transcript. Efficientpolyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one which is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BcII restriction fragment and directs both terminationand polyadenylation (Sambrook, supra, at 16.6-16.7). This 237 bpfragment is contained within a 671 bp BamHI/PstI restriction fragment.

The term “genetically engineered cell line,” refers to a cell line thatcontains heterologous DNA introduced into the cell line by means ofmolecular biological techniques (i.e., recombinant DNA technology).

The term “vector” as used herein, refers to a nucleotide sequencecomprising at least a promoter and a gene of interest. Such a gene ofinterest may encode an amino acid sequence for the purpose of expressingthe amino acid sequence (i.e., for example, a TSH receptor amino acidsequence). A vector has the capability of becoming integrated intoforeign DNA to form a stable transfected cell.

The term “stable transfection,” or “stably transfected,” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell.

The term “stable transfectant,” refers to a cell which has stablyintegrated foreign DNA into the genomic DNA.

The term “stable transfection” (or “stably transfected”), refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant,” refers to a cell whichhas stably integrated foreign DNA into the genomic DNA.

The term “RDluc” refers to an RD cell line having been stablytransfected with a luciferase gene. Further, RD-Rluc refers to a RD cellline having been stably transfected with a luciferase gene and thatdisplays an exogenous receptor (i.e., for example, a TSH receptorincluding but not limited to, a Mc4 receptor).

The term “CHOluc” refers to a CHO cell line having been stablytransfected with a luciferase gene. Further, CHO-Rluc refers to a CHOcell line having been stably transfected with a luciferase gene and thatdisplays an exogenous receptor (i.e., for example, a TSH receptorincluding but not limited to, a wild-type receptor). Alternatively,CHO-RMc4luc refers to a CHO cell line having been stably transfectedwith a luciferase gene that displays a chimeric receptor. (i.e., forexample, a TSH receptor that comprises amino acid sequences derived froma rat chorionic gonadotrophin receptor).

The term “selectable marker,” as used herein refers to the use of a genewhich encodes an enzymatic activity that confers resistance to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Selectable markers may be “dominant”; a dominant selectablemarker encodes an enzymatic activity which can be detected in anymammalian cell line. Examples of dominant selectable markers include thebacterial aminoglycoside 3′ phosphotransferase gene (also referred to asthe neo gene) which confers resistance to the drug G418 in mammaliancells, the bacterial hygromycin G phosphotransferase (hyg) gene whichconfers resistance to the antibiotic hygromycin and the bacterialxanthine-guanine phosphoribosyl transferase gene (also referred to asthe gpt gene) which confers the ability to grow in the presence ofmycophenolic acid. Other selectable markers are not dominant in thattheir use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene which is used in conjunction withtk cell lines, the CAD gene which is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withhprt cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook et al., supra at pp. 16.9-16.15.

The terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and“DNA encoding,” refer to the order or sequence of deoxyribonucleotidesalong a strand of deoxyribonucleic acid. The order of thesedeoxyribonucleotides determines the order of amino acids along thepolypeptide (protein) chain. The DNA sequence thus codes for the aminoacid sequence.

The terms “confluent” or “confluency” as used herein in reference to anadherent cell line define a condition wherein cells throughout a cultureare in contact with each other creating what appears to be a continuoussheet or “monolayer” of cells.

The terms “cytopathic effect” or “CPE” as used herein describe changesin cellular structure (i.e., a pathologic effect) resulting fromexternal agents such viruses. Common cytopathic effects include celldestruction, syncytia (i.e., fused giant cells) formation, cell roundingvacuole formation, and formation of inclusion bodies. CPE results fromactions of a virus on permissive cells that negatively affect theability of the permissive cellular host to preform its requiredfunctions to remain viable. In in vitro cell culture systems, CPE isevident when cells, as part of a confluent monolayer, show regions ofnon-confluence after contact with a specimen that contains a virus. Theobserved microscopic effect is generally focal in nature and the foci isinitiated by a single virion. However, depending upon viral load in thesample, CPE may be observed throughout the monolayer after a sufficientperiod of incubation. Cells demonstrating viral induced CPE usuallychange morphology to a rounded shape, and over a prolonged period oftime can die and be released form their anchorage points in themonolayer. When many cells reach the point of focal destruction, thearea is called a viral plaque, which appears as a hole in the monolayer.Cytopathic effects are readily discernable and distinguishable by thoseskilled in the art.

The abbreviation “ONPG,” representso-Nitrophenyl-13-D-Galactopyranoside. ONPG is a substrate for the enzymeβ-galactosidase (β-gal). The reaction between ONPG and (β-gal produces ayellow product which can be quantified spectrophotometrically at 405 nm.

The abbreviation “X-gal,” represents the chemical compound5-bromo-4-chloro-3-indolyl-(3-D-galactopyranoside, a substrate for theenzyme β-galactosidase. The reaction between X-gal and β-galactosidaseresults in the formation of a blue precipitate which is visuallydiscernable.

The term “hybriwix,” represents a product of Diagnostic Hybrids, Inc.,Athens, Ohio which allows for quantification of certain viral DNA in aninfected monolayer of cells by DNA hybridization. “DNA hybridization” isthe annealing of two complementary DNA molecules whose base sequencesmatch according to the rules of base pairing. DNA hybridization is usedto identify or quantify an unknown or “target” DNA by hybridization to aknown DNA or “probe.” The probe is typically labeled with a reportermolecule such as ¹²⁵I, a radioisotope which can be detected andquantified with a gamma counter.

The phrase “plaque reduction assay,” or “PRA,” as used herein describesa standard method used to determine efficacy of anti-viral drugs byenumerating a decrease in plaque formation in a cell monolayer exposedto a drug. A “plaque” is a defined area of “CPE.” It is usually theresult of infection of the cell monolayer with a single infectious viruswhich then replicates and spreads to adjacent cells of the monolayer. Aplaque may also be referred to as a “focus of viral infection.”

The term “permissive” as used herein describes the sequence ofinteractive events between a virus and its putative host cell. Theprocess begins with viral adsorption to the host cell surface and endswith release of infectious virions. A cell is “permissive” if it readilypermits the spread of virus to other cells. Many methods are availablefor the determination of the permissiveness of a given cell line,including but not limited to, plaque reduction assays, comparisons ofthe production and/or quantitation of viral proteins based on resultsobtained from gel electrophoresis, relative comparisons usinghybridization analysis to analyze DNA or RNA content, etc.

The term “susceptible,” as used herein describes the extent that apermissive or non-permissive host cell can adsorb and be penetrated by avirus. A cell line may be susceptible without being permissive in thatit can be penetrated but not release virions. A permissive cell linehowever must be susceptible.

The phrase “seed on,” as used herein describes the act of transferringan aqueous solution of suspended cells into a vessel containing cellsadhered to a surface, after which the vessel is stored for a sufficientperiod of time to allow the suspended cells or “seeds” to settle out bygravity and attach in a relatively uniform manner to the adhered cellsand become integrated into the final cell monolayer as a mixture. A“mixed cell monolayer,” results from the “seed on” process.

The phrase “seed in,” as used herein describes the mixing of two or moreaqueous solutions of suspended tissue culture cells, each cellsuspension having different cellular properties, and transfer of suchmixture of cells into a vessel which is stored for a sufficient periodof time to allow the suspended cells to settle out by gravity and attachin a relatively uniform manner such that the distribution of any singlecell type is indicative of the relative ratio of the cells in theoriginal mixture.

The term “starts,” as used herein refers to the reporter cells whichrepresent a primary infection of virus. The virus infects a reportercell (a genetically engineered cell) and induces the expression of thereporter gene. A reporter cell can be nonpermissive (i.e. permissivenessof the reporter cells is not required) and still produce starts.

The term “chimeric” as used herein, refers to any nucleic and/or aminoacid sequence containing portions from two or more different species. Aprotein may be chimeric if the primary amino acid sequence containsportions from two or more different species (i.e., for example, anhTSH/rLH-R or RMc4). A protein may also be chimeric if the primary aminoacids sequence contains portions from two or more different proteins,whether from the same species or different species. A protein may alsobe chimeric if the quaternary amino acid structure contains proteinsfrom two or more different species. Further, a nucleic acid may bechimeric if the primary nucleotide sequence contains portions from twoor more different species. A nucleic acid may also be chimeric if theprimary nucleotide sequence contains portions from two or more differentproteins, whether from the same species or different species.

The term, “glucocorticoid” as used herein, refers to any compound anycorticosteroid that increases gluconeogenesis, raising the concentrationof liver glycogen and blood glucose; the group includes, but is notlimited to, dexamethasone, prednisone, hydrocortisone, fluticasone,cortisol, cortisone, or corticosterone.

Graves' opthalmopathy, also known as Graves' thyroid-associated ordysthyroid “orbitopathy,” is an autoimmune inflammatory disorderaffecting the orbit of the eye. In mild disease, patients present witheyelid retraction. In moderate disease, patients present with myopathy.In more severe and active disease, mass effects and cicatricial changesoccur within the orbit. Disease may progress to a restrictive myopathywhich restricts eye movements and an optic neuropathy. With enlargementof the extraocular muscle at the orbital apex, the optic nerve is atrisk of compression. The orbital fat or the stretching of the nerve dueto increased orbital volume may also lead to optic nerve damage.

In some embodiments, serum samples are diluted (e.g. a series ofdilutions) and assayed in order to generate a serum dilution curve. Inone embodiment, the shape of serum dilution curves are compared togenerate a comparative reactivity profile (CRP). For example, in oneembodiment, the present invention contemplates generating a CRP bycomparing the shape of a serum dilution curves from a patient pre- (e.g.before initiation of therapy) and post- (after initiation of therapy)drug therapy/treatment. In this manner, one can compare the read out(e.g. luciferase activity) to determine reduction of thethyroid-stimulating immunoglobulin (TSI or TSAb) in the serum; bycomparing them, one can conclude whether the therapy is effective not.

In some embodiments, the present invention contemplates using the assaysand methods herein described to assess clinical activity, and/or theseverity of disease, and/or Graves' orbitopathy. In one embodiment, thepresent invention contemplates a method, comprising: a) providing: i) acell line comprising a stably transfected recombinant plasmid vectorencoding a chimeric human TSH receptor and a reporter gene; ii) a cellculture medium compatible with said cell line; and iii) a serum samplederived from a patient suspected of having Graves' disease; b)contacting the serum sample with the cell line and the medium (e.g. thecells are in the medium) under conditions such that a reporter geneemits a detectable signal upon induction by a TSH receptor-specificstimulating auto-antibody (in one embodiment, the signal intensityreflects the antibody concentration). In one embodiment, said chimericreceptor comprises a portion of a human chorionic gonadotropin receptor.In one embodiment, said method further comprises step c) measuring theactivity of the expressed reporter gene, wherein said activitycorrelates with clinical activity. In one embodiment, said methodfurther comprises step c) measuring the activity of the expressedreporter gene, wherein said activity correlates with the clinicalseverity of Graves' disease. In one embodiment, said clinical severityis assessed, measured or determined on the basis of diplopia, proptosis,visual acuity, ocular motility, optic neuropathy, or extra-ocular musclethickness as determined by either computed tomographic or magneticresonance imaging scans. In one embodiment, said clinical severity ismeasured on the basis of the NOSPECS score. In one embodiment, saidmethod further comprises step c) measuring the activity of the expressedreporter gene, wherein said activity correlates with Graves'orbitopathy. In one embodiment, the thyrotropin stimulating hormonereceptor autoantibody concentration correlates with clinical activity,and/or with the clinical severity of Graves' disease. The clinicalseverity may be assessed in a variety of ways. In one embodiment, theclinical severity is assessed on the basis of diplopia, proptosis,visual acuity, ocular motility, optic neuropathy, or extra-ocular musclethickness as determined by either computed tomographic or magneticresonance imaging scans. In one embodiment, said clinical severity ismeasured on the basis of the NOSPECS score. In one embodiment, thethyrotropin stimulating hormone receptor autoantibody concentrationcorrelates with Graves' orbitopathy. In one embodiment, said culturemedium contains a glucocorticoid. In one embodiment, said reporter geneis luciferase.

In one embodiment, diluted (e.g. with a buffer or other aqueoussolution) and undiluted serum samples are employed. In one embodiment,the present invention contemplates a method, comprising: a) providing:i) a cell line comprising a stably transfected recombinant plasmidvector encoding a chimeric human TSH receptor and a reporter gene; ii) acell culture medium compatible with said cell line; and iii) anundiluted serum sample and a diluted serum sample from a patientsuspected of having Graves' disease; b) contacting the undiluted serumsample and diluted serum sample with the cell line and the medium underconditions such that a reporter gene emits a detectable signal uponinduction by a TSH receptor-specific stimulating auto-antibody (in oneembodiment, the signal intensity reflects the antibody concentration).In one embodiment, said chimeric receptor comprises a portion of a humanchorionic gonadotropin receptor. In one embodiment, said method furthercomprises step c) comparing the activity of the expressed reporter genefrom said undiluted sample with said diluted sample. In one embodiment,said activity from undiluted and diluted serum demonstrates acomparative reactivity profile indicative of clinical activity. In oneembodiment, said activity from undiluted and diluted serum demonstratesa comparative reactivity profile indicative of severity of Graves'disease. In one embodiment, said activity from undiluted and dilutedserum demonstrates a comparative reactivity profile indicative forGraves' orbitopathy. In one embodiment, said culture medium contains aglucocorticoid. In one embodiment, said reporter gene is luciferase.

In one embodiment, the present invention contemplates testing pre- andpost-drug therapy serum samples. In one embodiment, the presentinvention contemplates a method, comprising: a) providing: i) a cellline comprising a stably transfected recombinant plasmid vector encodinga chimeric human TSH receptor and a reporter gene; ii) a cell culturemedium compatible with said cell line; iii) a first undiluted serumsample obtained from a patient having Graves' disease prior to beingtreated with drug therapy (pre-treatment); and iv) a second undilutedserum sample obtained from a patient having Graves' disease after beingtreated with drug therapy (post-treatment); b) diluting a portion ofsaid first serum to create a diluted first serum sample; and contactingsaid undiluted first serum sample and said diluted first serum samplewith the cell line and the medium under conditions such that a reportergene emits a detectable signal upon induction by a TSH receptor-specificstimulating auto-antibody (in one embodiment, the signal intensityreflects the antibody concentration). In one embodiment, the methodfurther comprises step d) diluting a portion of said second serum sampleto create a diluted second serum sample; and e) contacting the undilutedsecond serum sample and diluted second serum sample with the cell lineand the medium under conditions such that a reporter gene emits adetectable signal upon induction by a TSH receptor-specific stimulatingauto-antibody. In one embodiment, the method further comprises step f)comparing the activity of the expressed reporter gene from the first andsecond serum samples. In one embodiment, the comparative reactivityprofiles of the first and second serum samples serve as an indication ofpatient response to drug treatment. In one embodiment, said culturemedium contains a glucocorticoid. In one embodiment, said reporter geneis luciferase. It is not intended that the present invention be limitedby nature of the drug treatment. A variety of drugs and treatmentapproaches can be utilized. In one embodiment, the drug received by saidpatients is selected from the group consisting of anti-thyroid drugs,steroids, T4, and immunosuppressive drugs. In one embodiment, thethyrotropin stimulating hormone receptor auto-antibody concentration isused to monitor drug treatment (e.g. whether immunosuppressive drugtherapy is effective).

DETAILED DESCRIPTION

The present invention provides methods and compositions useful in thediagnosis of autoimmune diseases. In particular, the present inventionprovides methods and compositions for use in the diagnosis andmanagement of Graves' disease. For example, one composition comprises achimeric thyroid stimulating hormone receptor having improvedsensitivity and specificity for circulating thyroid stimulatingimmunoglobulin. Assays using such chimeric receptors can be optimized inthe presence of a glucocorticoid.

In addition, the present invention provides methods and compositions formonitoring the immune status and responses of individuals. Inparticular, the present invention finds use in monitoring the immuneresponses of vaccine recipients. The present invention further providesmethods and compositions for accelerating and enhancing the attachmentof viruses to cell surface receptors, providing increased sensitivity inassays to detect and quantitate viruses in samples.

I. Graves' Disease

Typically, the clinical picture of Graves' disease in young adults isvery easily recognized. The patients are more commonly female than male,and report symptoms including, but not limited to, sweating,palpitations, nervousness, irritability, insomnia, tremor, frequentstools, and weight loss in spite of a good appetite. Physicalexamination usually shows mild proptosis, stare, lid lag, a smooth,diffuse, non-tender goiter, tachycardia (especially after exercise) withloud heart sounds, and often a systolic murmur or left sternal borderscratch, tremor, onycholysis, and palmar erythema; often, a bruit isheard over the thyroid, and a cervical hum is almost always present. Inpatients with these symptoms, Graves' disease is readily recognized, andcan be confirmed with laboratory tests (See, Federman, Thyroid, in Daleand Federman (eds.), Scientific American Medicine, 3:1-6, ScientificAmerican, New York, N.Y., (1997).

Although the signs and symptoms described above can be troublesome,other symptoms of the disease can be more dangerous. One of the mostdisturbing symptoms is severe exopthalmos, accompanied byopthalmoplegia, follicular conjunctivitis, chemosis, and loss of vision.Additional symptoms include, but are not limited to, dermopathy,pretibial myxedema, clubbing, and in the most severe cases, acropachy.These signs and symptoms are indicative of a representative autoimmuneetiology of Graves' disease.

Despite the typical clinical picture of Graves' disease, methods areneeded to confirm the diagnosis, as well as provide prognosticindicators for management and treatment. In addition, in cases where thecause of hyperthyroidism is unclear, diagnostic test methods must beutilized to determine the etiology. Although in vivo methods such asradioactive-iodine uptake (RAIU) may be used in the diagnosis andmonitoring of patients with Graves' disease (See e.g., Baldet et al.,Acta Endocrinol. (Copenh) 116:7-12 (1987)), there are two basic groupsof in vitro assay systems developed for this purpose. One is dependentupon the measurement of some index of thyroid stimulation (e.g., cAMPgeneration) and the other assesses the ability of thyroid-stimulatingautoantibodies (TSAb) to inhibit the binding of radiolabelled thyroidstimulating hormone (TSH) to its receptor. These methods includebioassays and in vitro assays for TSAb. However, no widespreadapplication of methods to measure the thyroid-stimulating immunoglobulin(TSI or TSAb) in Graves' disease diagnosis has been reported. (See e.g.,Rapoport et al., J. Clin. Endocrinol. Metabol., 58:332-338 (1984)). Inaddition, it was recognized that in the sera of Graves' disease patientsthere is a heterogenous population of immunoglobulin G (IgG) moleculesthat recognize the thyroid hormone receptor (See e.g., Yokoyama et al.,J. Clin. Endocrinol. Metabol., 64:215-218 1987)). Further, therecognition that TSH-binding inhibition assays do not necessarilyreflect a thyroid-stimulating activity contributed to confusion inattempts to reach agreement on the clinical application of such assays(See e.g., McKenzie and Zakarija, J. Clin. Endocrinol. Metabol.,69:1093-1096 (1989)). Limitations in terms of sensitivity andspecificity were also problematic. Indeed, problems associated withavailable assay systems resulted in arguments that the measurement ofthyroid peroxidase antibodies is a sufficiently sensitive marker forunderlying thyroid autoimmunity (See, Botero and Brown, supra).

As indicated by Rapoport et al., the available assays that could beperformed easily, in a standardized manner, and for large numbers ofsamples had significant limitations in terms of sensitivity and/orspecificity, making these tests unreliable for clinical use. Theseproblems apply primarily to assays that measure the ability of TSI toinhibit the binding of radiolabelled TSH to human thyroid plasmamembranes (i.e., the assays do not measure TSI activity per se). Also,not all of the anti-TSH receptor antibodies are stimulatory. Rapoport etal. further indicate that assays using TSI stimulation of adenylatecyclase activity in human thyroid plasma membranes are seriously lackingin sensitivity. Some assays are unpractical for general clinical use,including those that rely upon the use of fresh human thyroid tissue,involve extremely difficult techniques with limited sample capacity, andare very laborious and/or uneconomical (See e.g., Rapoport et al,supra). The development of assays using cultured canine and porcinethyroid cells to measure the cAMP response to TSH were later adapted foruse with human thyroid cells which offered potentially superior results.In addition to the requirement for fresh thyroid cells in some of thesemethods (e.g., the methods discussed by Rapoport et al.), many alsorequired tedious and time-consuming sample preparation prior to assayingthe specimens. For example, some protocols require laborious andtime-consuming dialysis methods and/or precipitation of immunoglobulinsin the test sera with ammonium sulfate or polyethylene glycol (See e.g.,Rapoport et al., supra; and Kasagi et al., J. Clin. Endocrinol.Metabol., 62:855-862 (1986)).

In view of the problems encountered with these assay systems, othermethods were investigated in an effort to develop an assay that is easyto perform, reliable, sensitive, and specific for Graves' diseaseautoantibodies. For example, the use of bioassays to measure cAMPproduction rely upon the use of cells of non-human origin grown incontinuous culture or on human cells used as primary cultures or frozenin aliquots for use as needed. Problems with the use of human thyroidcells include the variability in responsiveness of surgically obtainedthyroid tissue. Thus, cells of nonhuman origin gained popularity,including the rat thyroid cell line (FRTL-5). This is a non-transformed,differentiated cell line that has been well-studied and characterized(See e.g., Bidey et al., J. Endocrinol., 105:7-15 (1985); andMichelangeli et al., Clin. Endocrinol., 40:645-652 (1994)). However, anumber of disadvantages make these cells less than ideal for Graves'disease assays. For example, the cells are slow growing and havefastidious growth requirements which include the need for TSH.Consequently, it is necessary to deprive the cells of TSH for at least 5days prior to assay in order to achieve a reasonable level ofsensitivity.

Subsequent development of cells such as the JP09 cells (Chinese hamsterovary cells transfected with a functional human TSH receptor) and othercell lines which stably express the human TSH receptor have greatlyimproved the assay systems available for the detection of Graves'disease autoantibodies. These cells have a TSH receptor that iscomparable to that of native thyrocytes and possess a functional signaltransduction system involving G-protein coupling, activation ofadenylate cyclase and cAMP generation in response to TSH and tothyroid-stimulating antibodies (TSAb) (See e.g., Michelangeli et al.,supra). These cells have been reported to be superior to FRTL-5 cells asthey provide similar diagnostic information, but are more sensitive,grow faster, have less fastidious growth requirements, and respond tounextracted sera, in comparison with FRTL-5 cells (Michelangeli et al.,supra; see also, Kakinuma et al., J. Clin. Endocrinol. Metabol.,82:212902134 (1997)). In addition, these methods are more rapid andreproducible, and perhaps more specific for detection of humanautoantibodies directed against the human receptor. Further, the assaysare easier and less cumbersome to perform than those using the FRTL-5cell line (See e.g., Vitti et al., J. Clin. Endocrinol. Metabol.,76:499-503 (1993)). However, these assays rely upon the use ofradioactivity (e.g., in radioimmunoassays) to detect and quantitate cAMPand are as a result, still cumbersome.

II. Diagnostic Assays for Graves Disease

Graves' disease is a thyroid disorder caused by an antibody-mediatedauto-immune reaction. In Graves' patients, the autoantibodiesrecognizing the TSHR (TRAbs) are heterogeneous, including mainly thyroidstimulating antibodies (TSAbs) and thyroid blocking antibodies (TBAbs.)TSAbs act as a TSH agonist causing hyperthyroidism while the TBAbsfunction as a TSH antagonist causing hypothyroidism. While TSAb and TBAbbind to different epitopes on the TSHR, TBAb binding can “neutralize”the stimulating effect of TSAb. When the TSAb binds to the TSHR, itinduces the cAMP signaling pathway, TBAb does not have this effect.

Currently, several bioassays are used to diagnose Graves' disease. TheKronus® Radio Receptor Assay (RRA) kit is used for determination ofTRAbs and detects both TSAbs and TBAbs but cannot distinguish betweenthe two. Diagnostic Hybrids Inc. (DHI) previously developed a Graves'diagnostic CHO-Luc cell line that detects the TRAbs in patient serum.This cell line co-expresses the wildtype TSH receptor gene and a fireflyluciferase gene which is driven by the human glycoprotein alpha subunitpromoter. This wild type TSHR has epitopes that bind TSAbs and TBAbs.Binding of TBAb to the receptor can modulate TSAbs' binding, resultingin lower stimulation by the TSAbs.

Thyroid-stimulating autoantibodies (TSAb) directed against the thyroidstimulating hormone (TSH) receptor are capable of stimulating thyroidadenylyl cyclase, the enzyme responsible for producing cyclic-adenosinemonophosphate (cAMP). These autoantibodies have been used as diagnosticmarkers for detection and identification of patients suffering fromGraves' disease, as these autoantibodies appear to be responsible forthe hyperthyroidism seen in patients with this disease. However, asdiscussed in more detail below, the methods commonly used to detect andmeasure these TSAbs are complex and time-consuming.

A. cAMP Detection

One method that measures TSAbs utilizes a rat thyroid cell line known as“FRTL-5.” This cell line, available from Interthyroid ResearchFoundation (Baltimore, Md.) expresses receptors that cross-react withhuman TSAbs. In the presence of TSAbs (i.e., for example, upon exposureof the cells to serum from a Graves' patient containing theseantibodies), FRTL-5 cells are stimulated to produce cAMP. This cAMP isthen measured in a portion of the lysed cells or the medium bathing thecells using a radioimmunoassay method. The FRTL-5 cells formed the basisfor the first successful bioassay for the autoantibodies that arepathognomonic of Graves' disease. U.S. Pat. No. 4,609,622 (hereinincorporated by reference).

B. FRTL-5 Cell Assays and Starvation Medium

A typical assay using FRTL-5 cells performed as described by Vitti etal. (Vitti et al., J. Clin. Endocrinol. Metabol., 76:499 (1993))involves seeding FRTL-5 cells in 96-well plates (30,000 cells/well) in aspecial complete medium containing 6 hormones (i.e., for example, a 6Hmedium) in addition to the normal growth constituents used in cellculture medium. After 2-3 days incubation in a 5% CO₂, humidified, 37°C. incubator (i.e., when the cells are confluent), the medium is changedto a “Starvation Medium,” which is deficient in TSH (thereby resultingin a 5H medium), wherein TSH is one of the 6 hormones in the 6H medium.The cells are then maintained for 4-5 days in the incubator with amedium change every 2-3 days. During this time the cells do not grow ormultiply. Subsequently, the cells may be used in a diagnostic assay.

C. Radiolabel Assays and Stimulation Medium

Early diagnostic methods for Graves' disease were performed by removingthe Starvation Medium and adding a Simulation Medium comprising aspecial low sodium chloride, high sucrose buffer (HBSS NaCl+222 mMsucrose; the formula for this buffer is: 0.0608 g/L KH₂PO₄, 0.144 g/LCaCl₂, 0.373 g/L KCl, 0.048 g/L MgSO₄, 0.097 g/L Na₂PHO₄, 1.0 g/LD-glucose, 76 g/L (i.e., 222 mM) sucrose, 4.77 g/L HEPES, and 10 g/LBSA; pH 7.2-7.4) containing a phosphodiesterase inhibitor (e.g., 0.5 mMmethylisobutylxanthine; IBMX), to prevent this enzyme from breaking downcAMP. Specially prepared samples of patient immunoglobulin (IgG),controls, and standards are added to the appropriate wells, usually intriplicate, and the plate is incubated in a 5% CO₂, humidified, 37° C.incubator for 2 hours. Following this incubation, 5-10 μl of the mediumare removed from each well and used in a radioimmunoassay system todetect the presence of cAMP. Typically this assay is run with about 6standards in duplicate, with patient and controls also run in duplicate.The assay usually requires an overnight incubation with about an hourrequired the next day for the separation of free, radiolabelled cAMPfrom antibody-bound, radiolabelled cAMP.

As the use of radioactivity and long preparation times are negativeaspects of the FRTL-5 assay, improved systems have been developed. Oneinvestigation involved the use of low salt conditions to increase thesensitivity of the assay system (See, Kosugi et al., Endocrinol.,125:410-417 (1989)). Additional improvements in the bioassay involved astrain of Chinese Hamster Ovary (“CHO”) cells transfected with a humanTSH receptor (“CHO-R”; See, Vitti et al., supra). This cell line offeredtwo major improvements over the FRTL assay. First, this method involvesthe use of human TSH receptors instead of rat TSH receptors which shouldprovide greater specificity and perhaps sensitivity for the detection ofTSAbs. Second, there is no requirement for the special 6H medium and 5Hmedium changes over a 6-8 day period, since the CHO-R cells grow well ona standard supplemented medium and can be used 1-3 days after seeding,depending on the density of the cell suspension used to inoculate thewells. In addition, comparative studies with FRTL-5 cells have shownthat the CHO-R cells may be more accurate in detecting Graves' TSAbs(See, Vitti et al.).

D. Luciferase Gene Assays Using CHO-Rluc Cell Lines

A further improvement was provided by the use of CHO-R cells designed toreadily assess the increased amounts of cAMP caused by TSI through theuse of a reporter gene (i.e., for example, luciferase) (Evans et al., J.Clin. Endocrinol. Metabol., 84:374 (1999)). Thus, with the introductionof this engineered cell line (i.e., CHO-Rluc), the complexity anddangers inherent in the use of radioactive compounds used in thepreviously developed radioimmunoassay for cAMP detection andquantitation are eliminated. With these cells, luciferase is measuredsimply by removing the medium from the cells, adding a lysis buffer,allowing 20-30 minutes for lysis to occur, removing a sample of thelysate, adding luciferase substrate and measuring light output over a 15second interval using a luminometer. However, as indicated in theExperimental section below, this method provides equivocal results andrequired further improvement.

In one embodiment, the present invention contemplates methods thatincorporate the advantages of a CHO-Rluc protocol, while providingadditional advantages in terms of reliability and reproducibility.Considerable development effort was dedicated to the development ofmethods of the present invention, including those that allow the use ofCHO-Rluc cells in luminometric assays using TSH and immunoglobulins fromuntreated Graves' disease patients.

The standard protocol originally used involved planting the CHO-Rluccells from a frozen stock, so as to seed at a concentration thatproduced confluent monolayers after 18-24 hours of incubation.Initially, the Growth Medium was removed and Stimulation Medium wasadded to the monolayers, to which a series of TSH standards (e.g., 0,10, 100, 1000 μIU TSH/ml), and patient IgG samples were added. As thisapproach yielded poor results, an overnight Starvation or conditioningperiod was tested.

A Starvation period resulted in improved results with lower backgroundvalues and appeared to produce good values for the TSH standards and thetest patient samples. An additional experimental option was also testedin which polyethylene glycol (PEG) was used to enhance antigen andantibody binding. In these experiments, PEG was added to the StimulationMedium.

In various experiments, different media formulations and combinationswere tested, as described in the Experimental section below. Forexample, starvation with the Stimulation Medium resulted in RLU/secvalues of (32,103) for the 0 μIU/ml TSH standard, −1,148 for the 10 μIUTSH/ml sample, 47,478 for the 1000 μIU TSH/ml sample, and 19,350 for IgGsample #13. In this, and the following discussions, the numbers inparentheses represent the 0 μIU TSH/ml value, which is subtracted fromthe values for the standards or samples to yield net values.

Starvation with standard HBSS resulted in RLU/sec values of (21,671) forthe 0 μIU/ml TSH control, 1,336 for the 10 μIU TSH/ml sample, 82,466 forthe 1000 μIU TSH/ml sample, and 39,082 for IgG sample #13. Starvationwith standard HBSS and 6% PEG in the Stimulation Medium resulted inRLU/sec values of (32,562) for the 0 μIU/ml TSH control, 5,980 for the10 μIU TSH/ml sample, 207,831 for the 1000 μIU 5 TSH/ml sample, and174,461 for IgG sample #13. Thus, starvation with standard HBSS yieldedhigher values for TSH and the Graves' disease samples, and theincorporation of PEG into the Stimulation Medium yielded even highervalues. These higher values appear to impart a higher level ofsensitivity in the methods of the present invention, as compared to theabove described methods. Nonetheless, the long duration of these assaysinvolving Starvation periods is disadvantageous. It was hypothesizedthat assay improvements that shortened the 3-4 days assay period mightalso improve assay sensitivity and accuracy.

E. Chimeric TSH Receptor Cell Lines

In one embodiment, the present invention contemplate recombinant celllines (i.e., for example, CHO and RD) that express a TSH/LH/TSH chimericreceptor (i.e., for example, RMc4) in combination with a fireflyluciferase gene. In one embodiment, the expression is driven by a humanglycoprotein alpha subunit promoter. Although it is not necessary tounderstand the mechanism of an invention, it is believed that by using achimeric receptor, binding of the blocking antibodies (i.e., forexample, TBAb) is either eliminated and/or reduced. In one embodiment, achimeric receptor comprises at least one genetic modification such thatonly a TSAb binding region is expressed. It is believed that therecombinant cell lines have increased specificity when compared toeither the CHO-Luc cells or KRONUS® assay.

III. Monitoring of Immune Response Development

As indicated above, the present invention also provides methods andcompositions for the monitoring of immune response development. Inparticular, the present invention provides methods and compositionssuitable for monitoring the response of individuals to vaccination.

In one embodiment, a pre-immune serum (i.e., serum collected prior toadministration of vaccine) may be used as a baseline for controlpurposes. Such serum would also be collected shortly followingvaccination (e.g., 1-2 weeks after vaccination), as well as periodicallyin the months following vaccination. The serum samples are then testedfor the presence and quantity of neutralizing antibodies.

In some embodiments, diagnostic assays are conducted to monitor theresponse to viral antigens. In such assays, cells such as ELVIS™(Diagnostic Hybrids, Athens, Ohio) are used in combination with apolyethylene glycol (PEG) solution of the present invention. In oneembodiment, PEG enhances the antigen-antibody reaction, therebyresulting in higher reactivity.

IV. TSI Detection in CHO-Mc4luc and RD-Mc4luc Cell Lines

In one embodiment, the present invention contemplates using geneticallyengineered Chinese Hamster Ovary (CHO) and/or human Rhabdomyosarcomacells (RD) for diagnosing Graves' disease and/or monitoring Graves'disease therapy.

Clinical laboratories currently utilize various cells and reactionbuffer for the detection and measurement of stimulating autoantibodiesspecific to Graves' disease in patient sera for identifying patientssuffering from this disease and monitoring their therapy. For example,cells comprising genetically modified CHO cells containing wild typehuman Thyroid Stimulating Hormone Receptor (TSHR) and the CRE-Lucreporter system are utilized by numerous laboratories. These cells,however, need one day for growth and one day for starvation which puts atime constraint on test results availability. On the third day, thepatient's serum specimens are incubated with the cells and reactionbuffer in order to detect the presence of the Graves' autoantibodies. Insome embodiments, the present invention contemplates methods that do notrequire these multi-day assay procedures. In one embodiment, theseshorter methods do not have a Starvation period incubation. Theadvantages of a quick, accurate, and sensitive assay to diagnose Grave'sdisease are explained more fully below.

In one embodiment, the present invention contemplates a method forimproving a thyroid stimulating immunoglobulin (TSI) detecting cell line(CHO-RLuc). In one embodiment, the cell line further comprises achimeric receptor. In one embodiment, the chimeric receptor comprises ahuman Thyroid Stimulating Hormone Receptor (TSHR) and a rat LuteinizingHormone (LH) (i.e., for example, a RMc4 receptor). Although it is notnecessary to understand the mechanism of an invention, it is believedthat a chimeric TSH receptor provides improved binding specificity forTSI such that a Starvation period in the diagnostic assay is notrequired.

In one embodiment, the present invention contemplates a method forexpressing the Mc4 chimeric receptor in the CHO cells and/or RD cells(or other mammalian cells). In one embodiment, the method furthercomprises using CRE-Luc as a reporter gene to detect TSI. In oneembodiment, the chimeric receptor provides greater specificity than awild-type receptor by preferentially binding to stimulatingautoantibodies (i.e., as opposed to blocking autoantibodies). In oneembodiment, the chimeric receptor provides greater sensitivity than awild-type receptor by preferentially binding to stimulatingautoantibodies (i.e., as opposed to blocking autoantibodies). In oneembodiment, the cell culture further comprises PEG. Although it is notnecessary to understand the mechanism of an invention, it is believedthat because Graves' patient sera can have both stimulating and blockingautoantibodies, the wild type TSH-R receptor will bind with bothantibodies equally. Further, it is believed that blocking autoantibodiescan moderate and suppress stimulating autoantibody activity.

These chimeric TSH-R receptors expressed in the disclosed cell linesoffer the following advantages over currently used cell lines:

1. The system results in a lower luciferase activity background leadingto higher Signal:Noise (S:N) or Signal:Background (S:B) ratios.

2. The cell lines do not need to be “starved” overnight, a requirementfor currently used cell lines in order to maximize the signal resultingfrom TSI binding. This change reduces the turn-around time from acurrent 3 day assay to a 2 day assay, which is very advantageous to thelaboratory, the physician, and the patient.

3. The assay is designed to measure stimulating antibodies, whereas thewild type TSH-R is responsive to both stimulating and blockingantibodies whereas this Mc4 chimeric receptor is responsive only tostimulating antibodies, thereby providing greater specificity for whatis being measured.

V. Chimeric TSH Receptor

In one embodiment, the present invention contemplates novel diagnosticcell lines that detect thyroid stimulating hormone receptor (TSH-R)autoantibody (i.e., for example, thyroid stimulating immunoglobulin;TSI) with high detection sensitivity and specificity. In one embodiment,the cell line comprises a recombinant Chinese Hamster Ovary cell (i.e.,for example, a CHO-K1 cell). In one embodiment, the cell line comprisesa Human Rhabdomyosarcoma (RD) cell.

In one embodiment, the present invention contemplates a vectorcomprising a nucleic acid sequence encoding a hTSH/rLH-R fusion protein(i.e., for example, RMc4) linked to a firefly luciferase reporter geneand in operable combination with a glycoprotein hormone alpha subunitpromoter. In one embodiment, a cell line is transfected with the vector.In one embodiment, the transfected cell line expresses a human TSH-R/ratLuteinizing hormone (LH) chimeric receptor (hTSH/rLH-R), underconditions such that the luciferase reporter signal is detected.

A. Chimera Construction

The identity of binding sites for TSH and thyroid stimulatingautoantibodies in relation to Graves' disease was initially examined byconstructing human/rat chimeric TSH-R constructs. A partial substitutionof the human TSH-R with the corresponding rat sequence resulted in thefollowing chimeric receptors: i) Mc1+2 substituting amino acid residues8-165; ii) Mc2 substituting amino acid residues 90-165; and iii) Mc4substituting amino acid residues 261-370. The data suggested that aminoacid residues 8-165 contain an epitope specific for thyroid stimulatingautoantibodies which are not the same as those required by TSH.Significant heterogeneity in the binding sites between idiopathicmyxedema thyroid stimulating antibodies, Graves' disease thyroidstimulating antibodies, and TSH was observed. Tahara et al.,“Immunoglobulins From Graves' Disease Patients Interact With DifferentSites On TSH Receptor/LH/CG Receptor Chimeras Than Either TSH OrImmunoglobulins From Idiopathic Myxedema Patients” Biochem Biophys ResComm 179:70-77 (1991).

Early studies demonstrated transfection and expression of chimeric TSHreceptors that included segments from rat TSH receptors and ratlutenizing hormone chorionic gonadotropin receptors. Various rat TSHamino acid sequences were substituted with the corresponding rat LH/GCsequences. The data demonstrated that amino acid residues 268-304 werenot critical for generating the cAMP response but did eliminate a TSHhigh affinity binding site. Akamizu et al., “Chimeric Studies Of TheExtracellular Domain Of The Rat Thyrotropin (TSH) Receptor: Amino Acids(268-304) In The TSH Receptor Are Involved In Ligand High AffinityBinding, But Not In TSH Receptor-Specific Signal Transduction” Endocr J40:363-372 (1993). The heterogeniety of anti-TSH receptor antibodies wasaddressed by comparing binding of: i) TSH-binding inhibitoryimmunoglobulin; ii) thyroid-stimulating antibody; and iii) thyroidblocking antibody using a chimeric human TSH receptor wherein amino acidresidues 90-165 of the human TSH receptor were substituted by equivalentamino acid residues from the lutenizing hormone chorionic gonadotropinreceptor. The binding data suggest that there might be two differenttypes of thyroid-stimulating antibodies, three different types ofTSH-binding inhibitory immunoglobulins, and one nonfunctional antibody.

Chimeric TSH receptors have been reported to detect and characterizevarious types of circulating antibodies suspected of having arelationship with Graves' disease. Such antibodies are believed toinclude, but are not limited to, stimulating autoantibodies that canactivate TSH-R and blocking autoantibodies that can block TSH-R bindingby either TSH or stimulating autoantibodies. For example, chimeras ofhuman TSH-R (hTSH-R) and lutenizing hormone human chorionic gonadotropinreceptor (LH-hCG-R) included an RMc4 chimera having amino acids 261-370of the hTSH-R substituted with equivalent residues from a human LH/CG-R.The ability of purified IgG samples from Graves' disease sera samples tostimulate cAMP production was measured by radioimmunoassay. Kung et al.,Epitope Mapping of TSH Receptor-Blocking Antibodies In Graves' DiseaseThat Appear During Pregnancy” J Clin Endocrinol Metab 86:3647-3653(2001).

The interactions between TSH stimulating and blocking autoantibodies wasaddressed by using two types of TSH-R chimera constructs. The firstchimera is designated Mc2 having human TSH-R amino acid residues 90-165substituted by equivalent residues from rat lutenizing hormone chorionicgonadotropin receptor. The second chimera is designated Mc1+2 havinghuman TSH-R amino acid residues 8-165 substituted by equivalent residuesfrom rat lutenizing hormone chorionic gonadotropin receptor. Evaluationof circulating autoantibodies in Graves' disease patients showed thatblocking autoantibodies do not strongly antagonize the action ofstimulating autoantibodies, but could be responsible for underestimatingstimulating autoantibody activities as measured by current CHO-hTSH-Rdiagnostic assay methods. Kim et al., “The Prevalance And ClinicalSignificance Of Blocking Thyrotropin Receptor Antibodies In UntreatedHyperthyroid Graves' Disease” Thyroid 10:579-586 (2000).

The DNA sequence of the chimeric hTSH/rLH-R receptor (RMc4) contains atotal of 2,324 base pairs and encodes 730 amino acids. FIG. 8. In thischimeric receptor, the human TSH-R region ranging from amino acid number262 to 335 was substituted with the corresponding 73 amino acids fromthe rat luteinizing hormone (LH) receptor

The sequence that drives the expression of the luciferase reporter is a236 nucleotide glycoprotein alpha subunit promoter, which contains acyclic AMP (cAMP) regulatory element (CRE) and was cloned by PCR. Thenucleotide sequence of the cloned promoter was determined by DNAsequencing and was confirmed by sequence comparison with Gene banksequence AF401991. An alignment of the cloned promoter with a GPHpromoter amplified by PCR from HEK cells indicate that the two sequencesare identical. FIG. 9.

C. Chimera Diagnostic Assay

The response of the CHO-RMc4luc, RD-RMc4luc and CHO-Rluc cell lines tonegative and positive TSI sera was then compared. The cells wereincubated with TSI negative and positive sera for three hours. Cellswere then lysed and luciferase activity was measured by a VeritasMicroplat Luminometer. The results indicated that both the CHO-RMc4lucand RD-RMc4luc cell lines had much higher detecting sensitivity whencompared to the CHO-Rluc cells. FIGS. 10A, 10B, 10C, 10D and 10E. Acomparison of the ratio of luciferase RLU from TSI positive sera to thenegative sera (ratio of S/N,) shows that CHO-RMc4luc and RD-RMc4luccells were 6-8 and 2.1-4 times more sensitive than CHO-Rluc cell line.FIGS. 10B and 10D, respectively. CHO-RMc4luc cells were about 1.3 to 3.5more sensitive than RD-RMc4luc cells. FIG. 10E. In addition, theCHO-RMc4luc had lower levels of induced luciferase activity thanCHO-Rluc when tested with TSI negative serum leading to lower backgroundand increased signal/negative (S/N) ratios. FIG. 10A. Furthermore, bothCHO-RMc4luc and RD-RMc4luc cell lines showed very low standard deviationvalues. FIG. 10A and FIG. 10C, respectively. A TSI positive serum,denoted #19, showed a high luciferase induction level on theCHO-RMc4luc, RD-RMc4luc and CHO-Rluc cell lines This serum was dilutedand tested on these cell lines to compare the sensitivities. FIG. 10E.

Further, detecting sensitivity between the CHO-Rluc, CHO-RMc4luc andRD-RMc4luc cell lines induced with a serially diluted TSI positive serumwas compared. For example, a TSI positive serum was serially diluted andincubated on the different cell lines for three hours. The RD-RMc4lucand CHO-Rluc cell lines showed linear responses of the ratio of S/N inthe serum dilution range between 1:2 and 1:8. However, the slope of thedose response (value) and hence, the detection sensitivity, forRD-RMc4luc was much higher than that of CHO-Rluc cell line. FIG. 11A.CHO-RMc4luc did not show a linear response of the ratio of S/N at no orlow serum dilutions. FIG. 11B. CHO-RMc4luc cells, however, did show alinear dose response of the ratio of S/N from the serum dilution rangingfrom 1:32 to 1:128. FIG. 11C. Note that the slope of the dose response(value) was even higher than that of RD-RMc4luc cell line. FIG. 11Aversus FIG. 11C.

The CHO-RMc4luc cell line was also compared to the CHO-Rluc cell linefor TSH sensitivity. The S/N ratio was derived from the luciferase assayusing CHO-Rluc, CHO-RMc4luc and RD-RMc4luc cell lines induced withrecombinant human TSH. Recombinant human TSH at various concentrationswas incubated with CHO-RMc4luc or CHO-Rluc cell lines for three hours.After incubation, the luciferase assays were performed. The resultsindicated that they both are able to detect TSH at a concentration aslow as 5 μIU/ml, but the detection sensitivity of CHO-RMc4luc was muchhigher than that of the CHO-Rluc cell line. FIG. 12.

CHO-RMc4luc and CHO-Rluc cell lines were also tested for theirspecificity using other anterior pituitary hormones including humanluteinizing hormone, LH,) human follicle stimulating hormone (hFSH) andhuman chorionic gonadotropin (hCG), all of which share a common alphasubunit. Neither cell line showed any cross activity with the testedhormones. Table 1.

TABLE 1 Specificity of CHO-Rluc and CHO-RMc4luc to human TSH and otherhormones. A. Luciferase S/N Ratio Comparision Of TSI Serum ToGonadotropin Hormones Ratio of Signal/Negative FSH LH HCG hTSH Positive(364 (455 (29.5 (76 TSI serum mIU/ml) mIU/ml) IU/ml) μIU/ml) CHO-Mc4luc9.4 0.6 0.9 0.8 19.4 CHO-Rluc 1.9 0.7 0.8 0.96 2.9 −TSI serum +TSI serumHormones B. CHO-RMc4luc Cell Line Results CHO-RMc4luc FSH (364 mIU/ml)RLU 1589 15069 1019 Ratio of S/N 9.5 0.6 LH (455 mIU/ml) RLU 1737 155651561 Ratio of S/N 9 0.9 hCG (29.5 IU/ml) RLU 1432 13491 1168 Ratio ofS/N 9.4 0.8 hTSH (76 μIU/ml) RLU 1284 12512 24880 Ratio of S/N 9.7 19.4C. CHO-Rluc Cell Line Results. CHO-Rluc FSH (364 mIU/ml) RLU 1052 1929728 Ration of S/N 1.8 0.7 LH (455 mIU/ml) RLU 1058 2011 835 Ration ofS/N 1.9 0.8 hCG (29.5 IU/ml) RLU 946 1976 912 Ration of S/N 2.1 0.96hTSH (76 μIU/ml) RLU 847 2291 2495 Ration of S/N 2.7 2.9

CHO-RMc4luc and CHO-Rluc cell lines were used to screen normal humansera to determine the distribution of the ratio of S/N derived from aluciferase assay. Comparisons of distribution of the S/N ratios derivedfrom luciferase assays on CHO-Rluc and CHO-RMc4luc cell lines inducedwith sera from 108 normal people were performed. All serum samples weretested in both CHO-RMc4luc and CHO-Rluc cell lines. A known normal serumwas used as a reference for calculating S/N ratios. The distribution ofCHO-RMc4luc cell line revealed a pattern very similar to that of theCHO-Rluc cell line. The mean of the CHO-Rluc cell was 1 and theCHO-RMc4luc was 0.88. The standard deviation of CHO-Rluc was 0.23 andCHO-Luc was 0.21. FIG. 13.

The responses of the CHO-RMc4luc, RD-RMc4luc and CHO-Rluc cell lines toclinical patient serum samples were compared. The ratio of S/N derivedfrom the luciferase assay on CHO-Rluc, CHO-RMc4luc and RD-RMc4 luc celllines induced with 12 clinical serum samples. Each of the 12 serumsamples was tested in the CHO-RMc4luc, RD-RMc4luc and CHO-Rluc celllines. Luciferase activities of these samples were compared to that froma known negative serum sample (negative reference). The results of thisstudy indicated that both the CHO-RMc4luc and RD-RMc4luc cell lines hadmuch higher detection sensitivity when compared to the CHO-Rluc cellline with the and CHO-RMc4luc cell line being the most sensitive cellline. FIG. 14.

VI. Chimeric Receptor Assay and the Starvation Pre-Conditioning Period

As discussed above, the CHO-RMc4luc cell line provides definiteadvantages in sensitivity and specificity over the currently usedCHO-Rluc cell lines. For example, the CHO-RMc4luc cells, uponstimulation by Graves' Disease antibodies, provide increased luciferaseresponses as measured by the Relative Light Units (RLU) output. Thisimprovement allows the elimination of the one day starvation step fromthe protocol.

The standard procedure used for the CHO-Rluc cell line includes astarvation period. The protocol for the Starvation format is to plantthe cells for Growth on day 1, Starve on day 2 and Stimulate and measureRLU on day 3. When the starvation period is eliminated, the “starve day2” step is eliminated and the final results can be reported to thephysician on day 2 instead of day 3, which is much more desirable. Thisnon-starved protocol provides a significant advantage to the user andphysician because labor for the user is significantly reduced and thephysician can have the results the next day, all with an assay of higheraccuracy than that provided by the CHO-Rluc protocol with its one day ofstarvation.

Starvation periods were used to increase the RLU or % separation betweenNormal and Graves' Positive sera. This RLU separation relates directlyto the accuracy of the assay. The effect of the Starvation period wastested using Graves' Positive and Normal serum specimens using the twodifferent cell lines, CHO-Rluc and CHO-RMc4luc. See, Table 8.

TABLE 8 Relative Effects Of Starvation Periods On CHO-Rluc AndCHO-RMc4luc Cells Cutoff Cutoff Cutoff 130% 140% 170% CHO-RLuc CHO-RMc4CHO-RMc4 Starved Non-Starved Starved Serum # (SRR) % RLU (SRR) % RLU(SRR) % RLU 26 118% 874 310.2% 13457 1023.3% 10162 Positive 27 149% 1105208.3% 8751  198.0% 1966 28 302% 2236 525.6% 22083 1291.5% 12825 29 504%3725 627.3% 27211 1912.2% 18988 30 380% 2811 481.4% 20885 1366.7% 776331 300% 2221 660.3% 27743 2345.7% 13323 32 571% 4226 525.9% 220961855.6% 10540 33 144% 1062 405.8% 17049 1495.9% 8497 34 208% 1537 150.5%6530  265.8% 1510 35 167% 1234 353.7% 15345  917.3% 5210 36 180% 1334574.2% 24909 2340.0% 13291 37 119% 878 187.5% 7878  388.9% 2209 38 114%846 231.9% 9745  468.6% 2662 39 104% 771 145.8% 6127  287.8% 3031 40137% 856 81.0% 3405  96.0% 1011 Ave. 1714 15548 7532  7 94% 504 39.2%1944    80% 525 Normal  8 90% 486 48.1% 2382    88% 658  9 89% 482 38.1%1887    78% 724 10 110% 591 37.8% 1872    79% 639 12 105% 566 51.1% 2290   90% 963 14 113% 611 43.1% 1930    99% 907 16 110% 591 35.9% 1608   75% 659 17 82% 443 36.8% 1648    85% 617 18 108% 582 40.5% 1816   77% 696 19 98% 542 42.6% 1908    84% 666 21 78% 431 84.4% 3781   101%613 23 90% 495 55.9% 2504    49% 587 24 125% 693 35.9% 1609    82% 42825 98% 541 50.8% 2274    70% 711 26 107% 591 45.7% 2049    75% 603 27104% 577 62.8% 2813    71% 647 28 109% 600 38.1% 1707    59% 613 29 96%532 48.2% 2021    83% 509 30 87% 481 39.5% 1655    87% 721 31 105% 48940.4% 1696    66% 769 32 75% 350 51.8% 2173    67% 589 33 89% 416 78.4%3289    62% 592 Ave. 527 2130 656 +RLU/−RLU 3.25X 7.3X 11.9X

The RLU values for Graves' Positive and Negative sera as obtained usingthe respective protocols for starved CHO-Rluc and CHO-RMc4luc cells, andnon-starved CHO-RMc4luc cells. The intended effect of starvation onCHO-RMc4luc cells was to decrease the background level of luciferaseactivity in the cells, thereby raising the ratio of RLU between Graves'Positive and Graves' Negative sera. For these sets of sera, the averageratio for Starved CHO-Rluc is 3.25×, for non-Starved CHO-RMc4luc is7.29× and for Starved CHO-RMc4luc cells is 11.5×.

Thus, the non-starved CHO-RMc4luc cells provide a greater than 2-foldincrease in luminosity (i.e., and therefore sensitivity) over thestarved CHO-Rluc cells. This provides distinct advantages of a protocollasting one day shorter and providing more accurate and sensitiveresults. The Serum:Reference Ratios (SRRs) compare the RLU values aspercentages and further confirm that the CHO-RMc4luc cells (whetherstarved or non-starved) provide an improved separation between the RLUsbetween Normal and Graves' Disease sera. The Cutoff values presented areapproximate and are indicative of Graves' Disease when the assayed valueis ≧ to the cutoff value of the particular protocol/cell line.

VII. Chimeric Receptor Assay Improvements with Glucocorticoids

Because the above data indicate that Starvation periods also provide animprovement of the accuracy and sensitivity of the CHO-RMc4luc cells,further investigations were then directed to develop superiorCHO-RMc4luc cell assays without a Starvation period.

One set of data was collected in accordance with Example 15, whereinCHO-RMc4luc cells underwent Starvation periods and then were incubatedwith various concentrations of dexamethasone (DEX). The data clearlyindicate that dexamethasone significantly improved the RLU intensityover and above that provided by a Starvation period alone. Table 9.

TABLE 9 Effect of Dexamethasone On Starved CHO-R-Mc4 Cells CHO-RMc4lucDEX Reference Serum uM 0 12. 25 40 50 100 pc* Test 1 151 141 132 126 122973 1601 Test 2 148 118 121 128 117 118 1277 Test 3 122 129 129 123 135120 1306 Avg 140 129 127 126 125 112 1395 S/B 9.9 DEX Patient #18 SerumuM 0 12.5 25 40 50 100 Test 1 2795 4710 5172 5039 4861 3542 Test 2 33935456 5151 5198 5298 3414 Test 3 3167 5330 5260 5038 4909 3521 Average3118 5165 5194 5092 5023 3492 S/B 22.2 39.8 40.6 40.4 40.1 31.1 % 100180 183 182 181 140 of S/B *Positive Control

The data show that dexamethasone reduces the Reference serum RLUreadings while at the same time greatly increasing Patient #18 RLUreadings. Overall, the presence of dexamethasone results in about an 80%increase in S/B ratios for the Patient #18 serum samples.

These observations provided a suggestion that an improved assay mayresult if dexamethasone is used in place of a Starvation period.Consequently, a comparison between cells exposed to a Starvation periodand cells only exposed to dexamethasone (40 μM) in the Growth Medium wasperformed. See Example 16. To compare the different protocols, the RLUresults for Graves' Positive and Graves' Negative serum were averagedtogether and their respective percentages above the Reference standardswere calculated. Table 10.

TABLE 10 Comparison Of Starved vs. Dexamethasone Treated CHO-rMc4 CellsCHO-Mc4 CHO-Luc Non-Starvation Protocol Protocol (Starvation) MC4 Mc4MC4 Mc4 w/Dex w/o Dex Luc w/Dex w/o Dex Luc Positive (n = 4) 12555 110231747 7978 4866 2486 Negative 1931 1885 1040 445 492 749 (n = 5)Reference 3436 5827 972 957 1129 691 (n = 1) Positive (n = 4) 365% 189%179% 834% 431% 360% Negative 56% 32% 107% 46% 44% 108% (n = 5) Reference100% 100% 100% 100% 100% 100% (n = 1)

The data demonstrate that non-starved CHO-RMc4luc cells withdexamethasone provides a more sensitive (and therefore more accurate)detection of circulating TSI's as compared to non-starved CHO-RMc4luccells without dexamethasone. For example, in the Graves' Positivepatient serums not subjected to a Starvation period, CHO-rMc4luc cellswith dexamethasone showed a 365% increase in luminescence (as relativeto the Reference) while CHO-rMc4 cells without dexamethasone showed a189% increase in luminescence.

The improvement in sensitivity and accuracy with dexamethasone is evenmore dramatic when comparing the Grave's Positive patient serums withthe Grave's Negative patient serums. For example, in the presence ofdexamethasone the difference between Grave's positive and Grave'snegative serums is 309% (i.e., for example, 365%-56%) but in the absenceof dexamethasone the difference between Grave's positive and Grave'snegative serums is 157% (i.e., for example, 189%-32%).

These results clearly show that dexamethasone provides a better assaysystem in terms of sensitivity due to a stronger luminescent signalstrength. Such an improvement results in improved testing accuracy andwhen combined with the RMc4luc testing platform, the present inventioncontemplates a diagnostic assay that is more rapid and accurate than anypreviously disclosed TSI antibody assay requiring a Starvation mediumperiod.

Further studies demonstrated that this effect was not limited todexamethasone but can be expected from most, if not all,glucocorticoids. For example, the data presented herein show that otherglucocorticoids also improve the sensitivity of the CHO-RMc4luc assay toprovide equivalent sensitivity in comparison with substitution for aStarvation medium period. Nonetheless, the presence of a glucocorticoidprovides the advantage that the assay can be performed in two days,rather than three days.

Alternative glucocorticoids (GCs) where compared to dexamethasone (40μM) on the basis of Relative Light Units (RLUs) and Serum ReferenceRatios expressed in percentages (SRRs %). For this purpose, all five (5)tested glucocorticoids show a generic effect in improving thesensitivity of the RMc4luc assay in the absence of a Starvation period.

The data was collected in accordance with the protocol outlined inExample 17. The data was calculated as the difference (Δ) in RLU valuesor SRR % values for each glucocorticoid (GC) concentration bysubtracting the RLU value or SRR % value for the Normal control at thatconcentration from the RLU value or SRR % value for the Positive controlat that concentration, respectively.

The data demonstrate that all four GCs improve signal intensity by atleast 6000 RLUs, wherein hydrocortisone and cortisone have signalintensities equivalent to dexamethasone. See, FIG. 17. When the data wascalculated as SRR %, however, it can be seen that dexamethasone improvessignal-to-noise ratio by approximately 2-fold when compared to all thealternative GCs. See, FIG. 18. Nonetheless, the data demonstrate thatany glucocorticoid can provide improvements in RMc4luc assay sensitivityand accuracy such that a Starvation medium period is not required.

VII. Kits

In yet other embodiments, the present invention provides kits forperforming Graves' disease diagnostic assays using chimeric TSHreceptors. The kits preferably include one or more containers containinga cell line-based diagnostic method of this invention. In someembodiments, the containers may contain a glucocorticoid including, butnot limited to, dexamethasone, cortisone, hydrocortisone, prednisone, orfluticasone. In some embodiments, the kits contain all of the componentsnecessary or sufficient for performing a Grave's disease diagnosticassay to detect circulating TSH autoantibodies in patient sera,including all controls, directions for performing assays, and anysoftware for analysis and presentation of results. In some embodiments,the kits contain vectors encoding chimeric TSH receptors capable oftransfecting cell lines. In some embodiments, the kits comprise allmaterials necessary or sufficient to perform diagnostic assays in asingle reaction and provide diagnostic, prognostic, or predictiveinformation (e.g., to a researcher or a clinician). For example, such akit might contain a cell line comprising a chimeric TSH receptor and aluciferase reporter system. In some embodiments, the kits comprise oneor more of a vector comprising a first nucleic acid sequence for an Mc4chimeric TSH receptor, a second nucleic acid sequence for aluciferin/luciferase reporter system, and a third nucleic acid sequencefor a promoter. Other embodiments also include buffers, controlreagents, detection devices, software, instructions, and TSHautoantibody standard preparations.

The kits may also optionally include appropriate systems (e.g. opaquecontainers) or stabilizers (e.g. antioxidants) to prevent degradation ofthe reagents by light or other adverse conditions. Each solution orcomposition may be contained in a vial or bottle and all vials held inclose confinement in a box for commercial sale.

The kits may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the reagents inthe diagnosis, detection, and/or treatment of Graves' disease. While theinstructional materials typically comprise written or printed materialsthey are not limited to such. Any medium capable of storing suchinstructions and communicating them to an end user is contemplated bythis invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); or L (liters); ml (milliliters); μl (microliters); μIU(micro International Units); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); ° C. (degrees Centigrade); sec. or s(second(s)); min. and m (minute(s)); MW (molecular weight); thyroidstimulating hormone or thyrotropin (TSH); bTSH (bovine TSH); TSI(thyroid stimulating immunoglobulin); TSAb (thyroid stimulatingantibodies); EDTA (ethylene diamine tetraacetic acid); RLU/sec (relativelight units per second); GM or PM (Growth Medium or Planting Medium); SM(Starvation Medium); HBSS (Hank's Balanced Salt Solution); EMEM (Eagle'sMinimum Essential Medium); FBS or FCS (fetal bovine serum or fetal calfserum); DMSO (dimethyl sulfoxide); CHO (Chinese hamster ovary cells);CHO-R (CHO cells transfected with the human TSH receptor; CHO-Rluc(CHO-R cells transfected with the cre-luciferase reporter gene complex);Oxoid (Oxoid, Basingstoke, England); BBL (Becton Diclinson MicrobiologySystems, Cockeysville, Me.)); DIFCO (Difco Laboratories, Detroit, Nil);U.S. Biochemical (U.S. Biochemical Corp., Cleveland, Ohio); Fisher(Fisher Scientific, Pittsburgh, Pa.); Sigma (Sigma Chemical Co., St.Louis, Mo.); ATCC (American Type Culture Collection, Rockville, Md.);LTI (Life Technologies, Rockville, Md.); and Promega (Promega Corp.,Madison, Wis.).

In the following methods, all solutions used in these methods weresterile (with the exception of TSH, controls, patient specimens) andtreated aseptically. All manipulations were conducted in a biosafetycabinet under aseptic conditions. Cell culture media (e.g., Ham's F-12,EMEM, etc.) were obtained from LTI, while additive reagents such asnon-essential amino acids were obtained from Sigma.

Freezer vials of cells should not be allowed to warm from their −80° C.(or lower) storage temperature until immediately prior to thawing anduse in the methods of the present invention, as cycling of thetemperature may result in viability losses. Because it containsdithiothreitol, which is unstable at room temperatures, the 5× celllysis solution should be removed from its −20° C. storage temperatureonly long enough to remove the required volume for preparation of the 1×solution. As it also contains dithiothreitol, reconstituted luciferasesubstrate solution should be kept frozen at −20° C. until just prior touse, at which time it may be removed and placed in a 25-37° C. waterbath to thaw and reach room temperature.

In general, when removing liquid from wells (e.g., microtiter plates,etc.), the liquid may be dumped from the wells into a receptacle in abiosafety hood. The residual liquid can be drained and removed byplacing the plate upside down on a sterile, absorbent wipe. Or, theliquid may be removed by aspiration using a fine tip on the aspirator.If aspiration is used, the plate is held at a steep angle so that theliquid does not overflow the wells, and the aspirator tip is directeddown the side of the well almost to the bottom to remove the liquid andonly leave minimal residue. However, care must be exercised in order toprevent disturbance of the cell monolayer, as the cells can be easilyremoved by the aspirator.

As indicated in the methods below, it is recommended that specimens,standards, and controls be run in triplicate. Because of the viscousnature of Solution 3 and the difficulty in achieving adequate mixing inthe wells, the best reproducibility was achieved when the totaltriplicate volume is +10% (33 μl) of these reagents is transferred tothe required triplicate volume +10% (330 μl) of Solution 3, thoroughlymixed, and 110 μl transferred to the triplicate wells.

In the preparation of cell monolayers (e.g., within the wells ofmicrotiter plates), it is preferred that the cells be distributed evenlywithin the wells. Thus, in order to avoid uneven cell distributions, thetransfer of cell suspensions into wells should be performed in avibration-free biosafety hood. After all of the wells in a plate havereceived cells, the plate is covered and carefully placed on a solid,vibration-free surface, for 30 minutes, to allow the cells to attachundisturbed, to the bottom of the wells. This helps ensure that an evendistribution of cells is present in each of the wells.

Example 1 Preparation of Cho-Rluc Cells for Testing

In these experiments, CHO-Rluc cells were prepared from W-25 CHO-R cellsfor use in the testing methods to detect TSI in Graves' diseasepatients. Pools of puromycin-resistant cells were obtained and testedfor light output in response to bovine TSH. Clones with the highestlight output were selected for use in the experiments described below.

CHO-Rluc cells were grown in cell culture flasks (e.g., T-225 flasks) ingrowth medium containing Ham's F-12 medium, 10% FBS (heated at 56° C.for 30 minutes to inactivate complement), 2 mM glutamine, and 1×non-essential amino acids. The flasks were incubated at 35-37° C., in ahumidified atmosphere, containing 5% carbon dioxide.

After the cell cultures reached confluence, the medium from each flaskwas aspirated, and the cell monolayers were washed with HBSS without Caand Mg⁺⁺. Then, 7 ml of a 0.25% trypsin/1 mM EDTA solution were added toeach flask, and allowed to react with the monolayers for approximately5-10 minutes at room temperature, in order to detach and disperse thecells in a nearly unicellular suspension. The cell suspensions were thencentrifuged for approximately 5 minutes at 300-400×g. The supernatantswere then removed and the pelleted cells resuspended in 8 ml of a mediumprepared by mixing 4 ml EMEM containing 1×HBSS and 20% FBS with 4 ml ofcryoprotective medium (EMEM containing 1×HBSS and 15% DMSO).

An aliquot of each cell suspension was then used to determine the numberof cells present in the suspension. This determination can beaccomplished using any method known in the art, including but notlimited to methods using a hemocytometer to determine the cell count.Thus, it is contemplated that any method can be used to determine thecell count in the suspensions. Based on the number of cells in thesuspension, the cells were aliquoted by volume to approximately 2×10⁶cells into standard freezer vials. The cells were then stored frozen at−90° C. for short-term storage. For long-term storage, the cells werestored in liquid nitrogen (about −200° C.).

Example 2 CHO-Rluc Assay Plate Preparation and Testing

In these experiments, CHO-Rluc cells prepared as described in Example 1were used in assays for diagnosis of Graves' disease. To prepare 24monolayers for testing, 24 wells in a 96-well microtiter plate werefirst treated by adding 50-100 μl 0.1% gelatin solution (Sigma) toenhance attachment of the cells to the bottom of the 24 wells chosen forthe test. Following incubation for approximately 1 minute at roomtemperature, the gelatin solution was removed from each of the wells byaspiration. It was noted that the gelatin can remain on the wells forlonger than one minute. The gelatin serves to coat the wells withcollagen, so that the cells attach more quickly to the wells and reachconfluence more rapidly. However, cells can be planted and grown toconfluence without gelatin and still perform well.

A freezer vial of CHO-Rluc cells produced as described in Example 1 wasrapidly thawed in a 37° C. water bath to provide approximately 0.4 mlcell suspension, which was well-mixed using a pipette. The cells werethen added to 2.5 ml GM (also referred to as “Planting Medium”),thoroughly mixed by vortexing for 1-2 seconds, and 100 μl aliquots ofthe cell suspension were added to each well, and the plates werecovered. It is preferable to produce an even distribution of cells ineach well. Thus, to avoid uneven cell distributions, the microtiterplate should be placed in a vibration-free hood for cell planting andattachment of cells to the walls of the microtiter plate. The plantedcells were then incubated at 35-37° C., in a humidified atmosphere,containing 5% CO₂, for approximately 20-24 hours, to allow the cells toform a nearly or completely confluent monolayer.

The GM was then aspirated from each well as completely as possible,being careful not to disturb the monolayers (i.e., confluent monolayersremain in the wells). The monolayers were rinsed with approximately 100μl Starvation Medium (HBSS containing Ca⁺⁺ (0.14 g/L) and Mg⁺⁺ (0.048g/L) per well. The Starvation Medium was aspirated and a fresh 100 μl ofStarvation Medium was then added to each well. It is important thatthese steps be conducted sufficiently rapidly that the cell monolayersdo not dry. The plates were then incubated overnight in a 35-37° C., 5%CO₂, humidified incubator. Following incubation, the Starvation Mediumwas aspirated from the wells, using care to avoid disturbing themonolayers. Then, approximately 100 μl Stimulation Medium were added toeach monolayer, again working quickly so that the monolayers did notdry.

Then, in an alternative method to that previously described, 10 μl ofpatient, control, and TSH standard solutions were added to theappropriate wells. The TSH standards and IgG samples were diluted withdiluent (i.e., HBSS-NaCl+222 mM sucrose). The TSH standards were testedat concentrations of 0, 10, 100, 1000, and 5000 μIU. Patient sampleswere diluted to a concentration of 10 mg protein/ml for use in theassay. As the Stimulation Medium is viscous, thorough mixing of thesuspensions was important. Adequacy of the mixing was ascertained bymicroscopic examination of the monolayers. The plates were incubated for4 hours at 35-37° C. in a 5% CO₂, humidified incubator. The medium wascarefully aspirated from each well and 150 μl lysis solution (Promega)was added to each well. The lysis solution contained 25 mMTris-phosphate, pH 7.8, 2 mM diaminocyclohexane tetraacetic acid (CDTA),2 mM dithiothreitol (DTT), 10% glycerol, and 1% Triton X-100. The plateswere then incubated for 30 minutes at room temperature, to allow themonolayers to lyse. Following lysis, each well was scraped and stirredusing a pipet tip. Then, 25 μl of lysate were removed from each well andplaced in a luminometer tube (12×75 mm, polypropylene), and 50 μl ofluciferase substrate (Promega) were then added. The tubes were vortexedfor 1-2 seconds and the RLU/sec values determined, using settings of 5seconds delay and 10 second read. To obtain average net values, theaverage of the “0 TSH” (i.e., the negative control) samples wassubtracted from all test average values.

Example 3 Preparation of IgG Samples

In these experiments, patients' IgG was prepared for testing in thepresent methods. Lyophilized IgG samples from 38 well-known andcharacterized, untreated Graves' disease patients were kindly providedby Dr. B. Y. Cho (Department of Internal Medicine, Seoul NationalUniversity, College of Medicine, Seoul, Korea). As most of the sampleshad been previously tested in standard methods using CHO-R and FRTL-5cells, these test results were known for 35 of these samples.

In preparation for lyophilization, the IgGs were affinity-purified usingprotein A-Sepharose CL-4B columns, as known in the art, and thendialyzed against 100 volumes of distilled water at 4° C. The dialysiswater was changed every 8 hours over a 2 day period. After removal ofdenatured protein by centrifugation at 1500×g for 15 minutes at 4° C.,the IgG was lyophilized and stored at −20° C. until used in theexperiments described herein.

In some experiments, purified untreated Graves' IgG was diluted innormal serum (euthyroid sera discussed in Example 7, below), and assayedusing the CHORluc assay described below.

Example 4 CHO-Rluc Assays

In these experiments, the performance of CHO-Rluc cells using the methoddescribed by Evans et al. (Evans et al., J. Clin. Endocrinol. Metabol.,84:374 (1999)) was evaluated. The media from the cell monolayers in the24 wells used in the 96-well microtiter plates prepared as described inExample 2 were aspirated and replaced with 100 μl Ham's F-12 mediumcontaining 10% charcoal-stripped calf serum (Sigma), and incubatedovernight at 35-37° C., in a humidified atmosphere containing 5% CO₂.

Then, 10 μl of bovine TSH standards diluted to a range of concentrations(e.g., 0 10, 100, and 1000 μIU) and Graves' IgG (dissolved to aconcentration of 10 mg protein/ml in charcoal-stripped calf serum) wereadded to respective quadruplicate wells. The suspension in each well wasmixed, and the plates were incubated for 4 hours at 35-37° C., in ahumidified atmosphere containing 5% CO₂. The medium was then aspiratedfrom each of the wells, and 150 μl of lysis buffer (Promega, asdescribed above) were added to each well. The plates were then incubatedat room temperature for 30 minutes to allow lysis of the cells in thewells. Then, 25 μl of each lysate were transferred to a 12×75polyethylene luminometer tube, to which 50 μl of luciferase substrate(Promega) were added immediately prior to mixing and reading in theluminometer at settings of 5 seconds delay and 10 second read. Theluminometer read out provided results as relative light units per second(RLU/sec). The negative or “zero” TSH standard value was subtracted fromeach of the readings. In one run, the average net value for the zeroμU/ml TSI standard was 68,011 RLU/sec, while the result for the samplecontaining 10 μIU/μl was 4031 RLU/sec, the sample containing 1000 μIUwas 222,801 RLU/sec, one Graves' IgG test sample was 384 RLUTsec (sample#1), and another Graves' IgG test sample was −3012 RLU/sec (sample #9).

The Graves' IgG sample #1 and sample #9 were previously assayed usingstandard FRTL-5 cells and a cAMP RIA assays. In the cAMP assay, valuesgreater than 153 with FRTL-5 cells are considered positive for thepresence of TSI. The cAMP value with FRTL-5 cells for sample #1 was 212,and the cAMP value for sample #9 was 803. The CHO-R values for thesesame samples (#1 and #9) were 116 and 1733, respectively, in an assaysystem where CHO-R values greater than 173 are considered to be positivefor Graves' disease. Thus, these results clearly indicate that there isa discrepancy between the results obtained using different cell linesfor the detection of Graves' disease. Indeed, the use of the Evans etal. method yielded negative results for both IgG samples, indicatingthat this system with CHO-Rluc is useless for detecting human TSI,despite the fact that the response to bovine TSH was very good.

Furthermore, during the development of the present invention (asdescribed below), it was determined that if the CHO-Rluc cells wereplanted in a medium containing charcoal-stripped calf serum for 24 hours(i.e., to reach confluence), the cells simply attached to the bottom ofthe wells, but did not multiply and become confluent during theincubation period, unlike the situation in which normal FBS was used.Thus, this surprising result indicates that the use of charcoal-strippedserum in the medium resulted in a starvation step for the cells,somewhat analogous to the incubation of FRTL-5 cells in 5H medium.

In some experiments purified, untreated Graves' IgG diluted in normalserum, were tested in the CHO-Rluc assay (with PEG). For IgG #10, (2mg/ml), the RLU/sec value was 131,461; for IgG #15 (2 mg/ml), theRLU/sec value was 180,327; for IgG #27 (5 mg/ml), the RLU/sec value was179,777; and for IgG#32 (5 mg/ml), the RLU/sec value was 112,627. Theseresults clearly show that the CHO-Rluc assay measures TSI in thepresence of serum.

Example 5 Development of Media Formulations

In view of the previously-described experiments, the effects ofdifferent media formulations were investigated for use with the CHO-Rluccells in the measurement of bovine TSH and human TSI. In theseexperiments, various media formulations were tested for the“starvation,” and “stimulation” steps in the CHO-Rluc assay, using bTSHstandards and IgG extracted from the sera of Graves' disease patients.

In these experiments, once the cell monolayers contained within thewells of 96-well microtiter plates (as described above), reachedconfluence, the Growth Medium was removed by aspiration and 100 μl ofStarvation Medium were added to each monolayer. The plates were thenincubated for 16-24 hours at 35-37° C., in a humidified atmospherecontaining 5% CO₂, to starve or condition the cells. The StarvationMedium was then aspirated from the wells.

To perform the assay, 10 μl of the patient specimen IgG, bTSH standards,and IgG controls (normal and Graves' disease sera), were added to themonolayers in triplicate. The suspensions were mixed within each well,and incubated under the above conditions for 4 hours. The liquid wasthen removed from each monolayer by aspiration, and 150 μl of lysisbuffer (Promega, as described above) were added to each well. The plateswere allowed to incubate at room temperature for 30 minutes to lyse thecells in the monolayers.

In order to measure the amount of cell stimulation caused by the TSHstandard or antibody to the TSH receptor, the luciferase in the celllysates was measured by adding 25 μl of lysate to a luminometer tube towhich 50 μl of substrate solution (Promega) were added. The suspensionswere mixed and then read in a luminometer with settings of a 5 seconddelay and a 10 second read, to determine the RLU for each sample.

In order to use the cells for TSI or TSH stimulation, the StarvationMedium was removed by aspiration, and 100 μl of the Stimulation Mediumwere added to each well. This Stimulation Medium was HBSS-NaCl+222 mMsucrose. The following Table 2 provides a comparison of the formulationsof HBSS-NaCl+222 mM sucrose and standard HBSS.

TABLE 2 HBSS Medium Formulation Comparisons HBSS--NaCl + 222 mM StandardComponent Sucrose (g/L) HBSS (g/L) CaCl₂ 0.144 g/L 0.14 g/L KCl 0.3730.400 KH₂PO₄ 0.060 0.060 MgSO₄ 0.048 0.048 Na₂HPO₄ 0.097 0.048 NaHCO₃0.00 0.35 NaCl 0.00 8.00 D-Glucose 1.00 1.00 Sucrose 76.00 0.00 HEPES4.77 0.00 Bovine Serum 10.00 0.00 Albumin

This Stimulation Medium formulation is a formulation that is commonlyused in the measurement of TSI in FRTL-5 and CHO-R cells.

The results of experiments to test various Starvation Mediumformulations are indicated in the following Table 3. In theseexperiments, the HBSS-NaCl+222 mM sucrose Stimulation Medium was used.As indicated in Table 3, the standard HBSS with 20 mM sucrose yieldedthe best signal to noise ratio (i.e., the lowest background and highestvalue for Graves' IgG).

TABLE 3 RLU/Sec Results for Various Media: Growth Versus StarvationRLU/Sec 10 μIU 1000 μIU #13 Medium 0 TSH TSH/ml TSH/ml IgG CHO GM^(a)(66,232) 782 265,195 5,144 CHO Char^(b) (50,638) 5,602 229,492 34,042HBSS--NaCl + 222 mM (32,289) 2,188 142,666 30,640 Sucrose^(c) StandardHBSS with 20 mM (27,139) 14,390 156,548 53,994 Sucrose^(c) ^(a)CHO GM isCHO Growth Medium containing 10% FBS. ^(b)CHO Char. is CHO Growth Mediumwith 10% charcoal-stripped calf serum. ^(c)A Starvation Medium

Example 6 Use of PEG

As PEG may be used in in vitro antigen/antibody reactions to assist orenhance the reaction rate, a trial was conducted in which PEG wasincorporated into the Stimulation Medium. As this compound may decreasethe off-rate or dissociation of the antigen/antibody complex, the use ofPEG in the methods of the present invention was investigated.

Preliminary results with 12% PEG-8000 (i.e., ave. MW 8,000) in HBSS-NaClsucrose, resulted in monolayers with increased spaces between the cells.To reduce this apparent osmotic stress, 6% PEG-8000 in HBSS-NaCl+111 mMsucrose was tested. In these experiments, the Starvation Medium yieldingthe best results (i.e., standard HBSS+20 mM sucrose) was used. Theresults are shown in Table 4, below.

TABLE 4 RLU/Sec Results for Stimulation Media With and Without PEGRLU/Sec 10 μIU 1000 μIU Stimulation Medium 0 TSH TSH/ml TSH/ml #13 IgGHBSS--NaCl + 222 mM (21,671) 1,336 82,466 39,082 Sucrose HBSS--NaCl +111 mM (32,562) 5,980 207,831 174,461 Sucrose + 6% PEG-8000

As indicated in Table 4, the incorporation of 6% PEG-8000 significantlyand substantially enhanced the luminescent signal from the CHO-Rluccells, in response to added bTSH, as well as Graves' IgG.

An additional experiment was conducted to determine the optimalconcentration of PEG-8000 to use in the Stimulation Medium. The netvalues for one Graves' sample (Graves' IgG #20), with an FRTL-5 cAMPvalue of 957, are shown in Table 5. As indicated in Table 5, 6% PEGyielded maximum signal for Graves' TSAb.

TABLE 5 RLU/Sec Results for Various PEG Concentrations % PEG InStimulation Medium Results 2% 4% 6% 8% 10% RLU/sec 15,566 52,259 87,90873,260 47,991

Subsequent experiments have shown that the Starvation Medium need notcontain 20 mM sucrose, as there is no statistically significantdifference in the results with or without it.

In addition, experiments were conducted to demonstrate that the assay ofthe present invention measures thyroid-stimulating immunoglobulin in adose-dependent manner. In these experiments, three Graves' disease IgGsamples (#6, #11, and #16) were tested. Serial 3-fold dilutions weremade using the Stimulation Medium containing 6% PEG-8000, and themethods described above. The results are shown in FIG. 1, which showsthe linearity of the dilutions. The IgG samples were prepared from 10mg/ml stocks, which were then tested undiluted, and serially diluted(3-fold dilutions) to 0.3333, 0.1111, 0.0371, 0.0123, and 0.0041dilutions (i.e., to yield 3.333 mg/ml, which was then diluted 3-fold toyield 1.111 mg/ml, etc.).

The FRTL-5 value for IgG sample #6 was 2080, while the FRTL-5 value forIgG sample #11 was 4453, and for IgG sample #16, the value was 830. Thefollowing Table 6 lists the results for each of these samples. Thecorrelation coefficients (r) were 0.857 for IgG sample #6, 0.858 forsample #11, and 0.995 for sample #16.

TABLE 6 Dose-Response (Dilution) Curves of Graves' IgG Specimens*Dilution Factor Sample 1 0.3333 0.1111 0.0371 0.0123 0.0041 IgG #6176,123 159,694 62,115 13,480 −6,628 −2,574 IgG #11 Not 368,373 324,143158,641 77,298 30,166 Done IgG #16 222,413 90,646 40,048 8,093 −1,705−691 *All values are reported as RLU/sec.

Example 7 Alternative Protocol Using PEG

In these experiments, alternative protocols using PEG were tested.First, freezer vials of CHO-Rluc cells were thawed, diluted in GrowthMedium (the contents of each cell vial were added to 2.5 ml medium), and100 μl of this cell suspension were added to each of the 24gelatin-coated wells of a 96-well microtiter plate, prepared asdescribed previously. The plates were incubated for 20-24 hours in a35-37° C., humidified incubator with an atmosphere containing 5% CO₂.This provided monolayers that were loosely confluent.

The Growth Medium was removed and the monolayers rinsed with 100 μl ofStarvation Medium (normal HBSS with Ca⁺⁺ and Mg⁺⁺), and a final 100 μlwere added to each monolayer before incubating overnight under theconditions described above. Following incubation, the Starvation Mediumwas removed and 100 μl of Stimulation Medium containing 6% PEG (i.e., asdescribed above) were added to each monolayer. Then, 10 μl of each ofthe standards and samples were placed into the wells (in triplicate).While other volumes were tested (e.g., 25 μl, 50 μl, and 75 μl), thevalues obtained were substantially equivalent to those obtained with 10μl volumes. Thus, the smaller volume was used in order to conserve thesamples and reagents, and to minimize the concentration of potentiallyinterfering substances present in some serum samples.

The well contents were mixed and the monolayers incubated as describedabove for 4 hours (i.e., a stimulation step). The medium was removedfrom each well, and 150 μl of lysis solution (as described above) wereadded to each well. The monolayers were allowed to stand at roomtemperature for 30 minutes for lysis to occur. Then, 25 μl of eachlysate were added to individual luminometer tubes. Fifty microliters ofluciferase substrate (as described above) were added to each tube, thecontents mixed, and the tubes immediately read in a luminometer withsettings of 5 seconds delay and a 10 second read time.

In an experiment to determine the normal range of euthyroid sera, 24specimens obtained from a reference laboratory were run using theCHO-Rluc assay as described above. The sera were euthyroid in that noneof the samples were submitted for thyroid testing. The mean (55,334RLU/sec) and standard deviation (1 SD 7,434 RLU/sec) were calculated forthese 24 euthyroid samples. The results are shown in FIG. 7. The SDvalue was then multiplied by three, which yielded a cut-off for normal,non-Graves' disease values of 77,636 RLU/sec. This cut-offencompasses >99% of the normal population; values greater than this wereconsidered to be TSI positive.

In a separate set of experiments, a group of 17 patient specimens whichpreviously been tested by a commercial esoteric testing laboratory usingcAMP RIA and FRTL-5 cells for TSI, were tested using the CHO-Rluc cellswith the above procedure. The FRTL-5 test results indicated 16 of thepatient specimens were negative for TSI (i.e., only one was positive).The single positive specimen identified by the FRTL-5/cAMP assay (258%or 1.98× the cut-off, where the assay cut-off was 130%), was likewisepositive by the CHO-Rluc assay (190,691 RLU/sec) based on a 2.45×cut-off of 77,636 RLU/sec, as shown in FIG. 7. The CHO-Rluc values ofthe 16 patient specimens which were negative (i.e., normal) by theFRTL-5/cAMP assay were found to be in good agreement with the 24 normalsera used to establish the normal range for the assay. See, FIG. 7.

Example 8 Comparison of CHO-Rluc Method and Standard Methods

In these experiments, the methods of the present invention utilizingStimulation Medium containing 6% PEG-8000 were compared with methodsusing the standard HBSS-containing Starvation Medium and StimulationMedium, to obtain luciferase values for 35 of the untreated Graves'disease IgG specimens obtained from Dr. Cho. The cAMP values obtained byDr. Cho with FRTL-5 and CHO-R cells using the same IgG samples as usedin methods of the present invention are shown in comparison with theCHO-R luciferase results in FIGS. 2, 3 and 4. FIG. 5 shows the linearityof luciferase response to bTSH.

FIG. 2 provides a comparison of CHO-Rluc luciferase results with theFRTL-5 cAMP results. This Figure indicates that the correlation betweenthese methods is quite good. FIG. 3 provides a comparison of CHO-Rlucluciferase results with CHO-R cAMP results. The CHO-R CAMP cut-off valuewas 173. Values below this cutoff were as follows (CHOluc RLU/sec): 110(219,913), 113 (14,434), 116 (25,373), 152 (84,493), 156 (7576), and 161(61,321). As indicated in this Figure, the range of CHO-R cAMP resultsis relatively narrow, as compared with the CHO-Rluc values. This is alsoshown in FIG. 4, which provides a comparison of CHO-R cAMP results withFRTL-5 cAMP results. The CHO-R value was 173. The FRTL-5 cut-off valuewas 153. Values below cutoff were as follows (FRTL-5 values): 110 (830),113 (283), 116 (212), 152 (1100), 156 (388), and 161 (335). The average+/−SD values for the IgG Control (ICN), for the tests shown in FIG. 2were 472+/−4015 (n=8).

FIG. 5 shows the linearity of the response to bTSH of the CHO-Rluccells. In these experiments, dilutions of bTSH were tested. The RLU/secvalues obtained are shown in Table 7, below.

TABLE 7 Results for bTSH Dilutions μIU TSH · ml Results 0 10 25 50 75100 RLU/sec 0 5,921 20,227 34,426 54,396 62,206

It is contemplated that this linearity and sensitivity of response tobTSH will prove useful in the detection of blocking antibodies to theTSH receptor (e.g., those autoantibodies in patents with atrophicthyroiditis and Hashimoto's thyroiditis which block the TSH receptor,thereby preventing thyroid hormone production and release resulting inhypothyroidism). This Figure also provides at least a partialexplanation of why the CHO-R cell line is not as sensitive to TSI fromGraves' disease patients sera as the FRTL-5 cell line. In these results,the correlation coefficient (r) was 0.9925. The three S.D. (standarddeviations) sensitivity was 1.3 μIU TSH/ml.

Example 9 Monitoring of Immune Responses

In these experiments, the immune response of vaccine recipients ismeasured and monitored. Although it is not intended that the presentinvention be so limited, this Example describes the monitoring of asubject's immune response to herpes simplex (HSV) vaccine.

Prior to administration of vaccine, a serum sample (i.e., preimmuneserum) is collected from the subject for use as a baseline or control,and stored frozen until testing. Serum samples are also collected atperiodic intervals following administration of the vaccine (e.g., 1-2weeks, 1 month, 2 months post-vaccination, etc.). The sera are thawed asnecessary, and used in an assay to determine the presence and quantity(i.e., titer) of neutralizing antibodies. Sera are serially diluted andmixed with known quantities of HSV. These samples are diluted indilutent comprising Eagle's MEM with HBSS containing 2 mM glutamine, 2%FBS, and PEG (e.g., 6% PEG 8000). However, it is also contemplated thatother diluents will find use in the present method, including diluentscontaining different concentrations and types of PEG, as appropriate forthe virus and assay system used). These samples are added to cellmonolayers containing cells capable of producing an enzyme such as(3-galactosidase upon infection with HSV (e.g., ELVIS™ cells, DiagnosticHybrids). Following overnight incubation under standard cell cultureconditions, the monolayers are lysed and the enzyme activity is measuredusing chromogenic or luminogenic methods.

A positive response to the vaccine is indicated by the lowest dilutionof postvaccination serum which neutralizes HSV in the sample (i.e., asindicated by a low OD. or luminescence value, in comparison with thepreimmune control).

In summary, the present invention provides numerous advances andadvantages over the prior art, including the avoidance of radioactivity,in combination with the advantages of ease of use, reliability,sensitivity, specificity, cost-effectiveness, and reproducibility.

Example 10 Construction Chimeric TSH-R Plasmids

This example presents one embodiment of constructing a cell linecomprising a chimeric TSH-R receptor for detecting Graves' diseaseautoantibodies.

Plasmid Construction

A plasmid comprising a first nucleic acid sequences encoding a TSH-Rchimeric receptor and a second nucleic acid sequence encoding a neomycinresistant gene was ligated to a luciferase gene and a glycoproteinhormone alpha subunit promoter.

Human Glycoprotein Alpha Subunit Promoter Cloning

Chromosomal DNA was isolated from human embryonic kidney cells using aQIAGEN RNA/DNA kit (QIAGEN Cat#14123.) Glycoprotein alpha subunitpromoter fragments were amplified by PCR using the isolated chromosomalDNA as the PCR template and the 2 pairs of oligo-nucleotide primersshown below:

(SEQ ID NO: 1) 5′PCR primer:5′-GAGCTC ATG TGT ATG GCT CAA TAA AAT TAC GTA  CAA AGT GAC AGC-3′(SEQ ID NO: 2) 3′ PCR primer:5′-AGATCT TCG TCT TAT GAG TTC TCA GTA ACT GCA  GTA TAA TGA AGT-3′.

A Sac I restriction site was added to the 5′ end of the 5′ PCR primerwhile a Bgl II restriction site was added to the 5′ end of the 3′ PCRprimer (both shown as underlined sequence). For PCR amplification, BDAdvantage 2 Polymerase Mix (BD Bioscience Palo Alto Calif.) was used andPCR reactions were performed in a thermal cycler (Eppendorf MastercyclerPersonal, Germen.). Forty cycles were carried out at 94° C. for 30seconds to denature the DNA. Samples were then annealed to the primersin the thermalcycler at 63° C. for 30 seconds, and the extension wasinduced at 68° C. for 1 minute 30 seconds per cycle. Two amplicons (1.2kb and 0.6 kb) were cloned into the plasmid vector pcDNA2.1 (Invitrogen,Carlsbad, Calif.) and sequenced using the BigDye Terminator v3.0 CycleSequencing method on an ABI 377 automated sequencer (Davis SequencingInc.).

Construction of Plasmid pGHP/Luc

The human glycoprotein alpha subunit promoter was isolated from vectorpcDNA2.1 by restriction cleavage with Sac I and Bgl II. The resulting316 bp fragment was then subcloned into the Sac I/Bgl II site of thepGL2 enhancer plasmid (Promega, Madison, Wis.) for construction of aplasmid named pGHP/Luc.

Construction of Plasmid pMc4-neo

The neomycin resistance gene for antibiotic selection (positive cloneselection) was isolated from vector pMC 1 (Stratagene Cedar Creek, Tex.)with restriction enzymes of XhoI and HicII. The resulting fragment wasthen subcloned into the XbaI site of plasmid pMc4 that contains theTSHR/LH chimeric receptor driven by the SV40 promoter (from Dr. LeonardKohn.) The final plasmid was named pMc4-neo.

Construction of Plasmid pMc4-Bsd

The antibiotic selection gene Blastocidin, isolated from vector pCMV/Bsd(Invitrogen, Carlsbad, Calif.) with restriction enzymes XhoI and XbaI,was subcloned into the XbaI site of plasmid pMc4 which contains theTSHR/LH chimeric receptor. Tahara et al., “Immunoglobulins From Graves'Disease Patients Interact With Different Sites On TSH Receptor/LH/CGReceptor Chimeras Than Either TSH Or immunoglobulins From IdiopathicMyxedema Patients” Biochem Biophys Res Comm 179:70-77 (1991). The finalplasmid was named pMc4-Bsd.

Construction of Plasmid pMc4-GHP/Luc

The human glycoprotein alpha subunit promoter, with a firefly luciferasereporter gene, was isolated from vector pMc4/Luc following restrictioncleavage by SmaI and AccI. The isolated DNA fragment was then subclonedinto the PfoI site of pMc4-neo plasmid. The final plasmid was namedpMV4-GHP/Luc.

Example 11 Mammalian Cell Selection

Seven different mammalian cell lines were tested to select the cell linethat had the lowest cyclic AMP basal level and highest potentialinducible levels. The results demonstrated that the CHO and RD cellsshowed the lowest cyclic AMP basal activity and the highest potentialinducible level. This empirical research approach maximizes the assaysensitivity by proper selection of cell culture type. For example, alower cyclic AMP basal level increases the sensitivity of the luciferaseassay. Also, the highest induced expression of cyclic AMP improves theaccuracy of the luciferase assay.

Example 12 Transfection/Selection of a CHO Cell Line with Chimeric TSH-RPlasmid

This example describes the permanent transfection of CHO cells.

Chinese Hamster Ovary cell line (CHO-K1; ATCC Number: CCL-61, ManassasVa.) was transfected with a linearized (Xmn1) pMc4-GPH/Luciferaseplasmid using HyFect® (Denville Scientific, Metuchen, N.J.) according tothe manufacturer's instructions. The CHO-K1 cells were then grown inHam's F12 Medium with 10% (v/v) fetal bovine serum and nine essentialamino acids at 37° C. in a humidified atmosphere containing 5% CO₂.Twenty four hours after the transfection, the cells were combined andplanted into a 96 well plate and selected with 0.5 mg/ml G418 in Ham'sF12 Medium with 10% FBS.

Example 13 Transfection/Selection of an RD Cell Line with Chimeric TSH-RPlasmid

This example describes the transfection of a Human Rhabdomyosarcoma (RD)(ATCC Number: CCL-136.) cell line with two plasmids, pGHP/Luc andpMc4-Bsd, in series to facilitate detection.

RD cells were transfected with the linearized pGPH/Luc (Sca1) plasmidusing HyFect (Denville Scientific, Metuchen, N.J.) according to themanufacturer's instructions. The cells were selected with 0.5 mg/ml ofneomycin. The optimal clone from this transfection and selection wasthen transfected with the linearized plasmid pMc4-Bsd. Aftertransfection, the cells were selected with both neomycin (0.5 mg/ml) andblasticidin (5 μg/ml.) to produce the final RD recombinant cell line.

All CHO and RD antibiotic resistant clones were tested with TSI-positiveand normal serum to select the clone which can be used for the detectionof TSI. The TSI induction positive clone was subjected to the limitingdilution cloning to further select a single clone.

The final clones have the ability to diagnose Graves' disease and/ormonitor the drug treatment of patients with Graves' disease with highersensitivity than the current product on the market. These cell linesshow good stability, having been passaged more than ten times, andcontinue to show very similar performance characteristics.

Example 14 Induction of Cell Lines with TSI Containing Serum

CHO cells from freezer vials were diluted and grown in growth media(Ham's F12 Medium with 10 (v/v) % fetal bovine serum and nine essentialamino acids) for 16 hours at 37° C. and 5% CO₂. After 16 hours the mediawas removed and the CHO cells were rinsed and refed with 100 μl/well“starvation” HBSS medium. The CHO cells were then incubated for 22-24hours. Following incubation the media was removed and CHO cells wererinsed and refed with 100 μl/well reaction buffer. The CHO cells werethen induced with a 1:11 dilution of patient serum in reaction buffercontaining BSA, PEG, sucrose, glucose, and salts (Diagnostic HybridsCatalog number 40-300500;) for 4 hours at 37° C. and 5% CO₂.

RD cells were grown in Eagles Minimal Essential Medium (EMEM) with 10(v/v) % fetal bovine serums at 37° C. and 5% CO₂ for 16-24 hours. RDcells were then directly induced with patient serum in reaction buffer(Diagnostic Hybrids Catalog number 40-300500) for 4 hours at 37° C. and5% CO₂.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled indiagnostics, cell culture, and/or related fields are intended to bewithin the scope of the following claims.

Example 15 Effect of Dexamethasone on Starved CHO-RMc4luc Cells

CHO-Rluc (041307A) and CHO-RMc4luc (062707, P8, 3e6/ml) cells were eachbrought up in 5 ml CHO growth media. From this stock of cells enough wastaken out and aliquoted into 6 tubes so that there would be 66,666cells/well for CHO-Rluc assay and 50,000 cells/well for the CHO-RMc4lucassay for each dexamethasone concentration (100, 50, 40, 25, 12.5, and 0μM). The dexamethasone stock was 500 μM in CHO-growth media. Alldexamethasone was made fresh. These vials were spun down and the cellswere brought up in the different concentrations of dexamethasone in CHOgrowth media.

Cells were then grown for 24 hours, rinsed with starvation media, andthen incubated in dexamethasone-containing starvation media thatcontained the same for 24 hours. The cells were then rinsed withreaction buffer and serum (diluted 1:11) was added to 100 μl of reactionbuffer already in the well. Serums used were: i) reference 121506R; ii)PC 121506P; and iii) patient #18 TSI serum. Reference and Patient #18serums were both treated the same and incubated with all 6concentrations of dexamethasone.

After 4 hours of induction the plates were lysed with 75 ul of BrightGlo®, lysed for 5 minutes, and then read on a Veritas luminometer.

Example 16 Comparison of Starvation Periods to Dexamethasone Treatment

Nine (9) human serum samples were prepared where four (4) samples wereknown positive for Grave's Disease and five (5) samples were knownnegative for Grave's disease. Each of the nine samples was tested ineach condition of both the Non-Starvation/Dexamethasone Protocol and theStarvation Protocol.

1. Non-Starvation/Dexamethasone Protocol

Three cell plates were analyzed using a CHO-RMc4 protocol. Plate 1:CHO-Mc4luc cells (4e6 cells/plate)+Growth Medium containing 40 μMdexamethasone in CHO-Mc4 Reaction Buffer. Plate 2: CHO-RMc4luc cells(4e6 cells/plate)+Growth Medium without 40 μM dexamethasone in CHO-Mc4Reaction Buffer. Plate 3: CHO-Rluc cells (4e6 cells/Plate)+Growth Mediumwithout 40 μM dexamethasone in CHO-Rluc Reaction Buffer

Each plate underwent a 16 hour growth period, a 3 hour induction period,and a 10 minute lysis period.

2. Starvation Protocol

Three plates were analyzed using a CHO-R protocol. Plate 1: CHO-RMc4cells (4e6 cells/plate)+Growth Medium containing 40 μM dexamethasone inCHO-Mc4 Reaction Buffer. Plate 2: CHO-RMc4luc cells (4e6cells/plate)+Growth Medium without 40 μM dexamethasone in CHO-Mc4Reaction Buffer. Plate 3: CHO-Rluc cells (4e6 cells/Plate)+Growth Mediumwithout 40 μM dexamethasone in CHO-Rluc Reaction Buffer. The samplearrangement for each protocol was configured identically. See, FIG. 16.

Each plate underwent a 24 hour growth period, a 24 hour starvationperiod, a 4 hour induction period, and a 5 minute lysis period. Theluminosity for each sample was measured using a Veritas luminometer.

Example 17 Comparison of Alternate Glucocortoids with Dexamethasone

This example provides data showing that improved sensitivity of aCHO-RMc4 assay is not limited to the substitution of a Starvation mediumperiod with dexamethasone (Dex) in accordance with Example 16. Thesedata demonstrate that four alternative glucocorticoids (GCs) haveequivalent effects in improving signal intensity.

Four GCs were examined in this study: i) Prednisone (Sigma); ii)Hydrocortisone (Sigma); iii) Fluticasone Propionate (Sigma); and iv)Cortisone (Sigma). A stock concentration (100 mM) of each GC was made inDMSO. 1:10 (10 mM) and 1:100 (1 mM) dilutions, in DMSO, were then madefrom the 100 mM stock. All DMSO/GC stocks were clear with no visibleprecipitate.

Growth Media containing different concentrations of each GC were made asfollows: i) 100 μM GC Medium: 5 μL of 100 mM GC Stock+5 mL SR097 (w/oDex); ii) 50 μM GC Medium: 2.5 μL of 100 mM GC Stock+5 mL SR097 (w/oDex); iii) 10 μM GC Medium: 5 μL of 10 mM GC Stock+5 mL SR097 (w/o Dex);iii) 1 μM GC Medium: 5 μL of 1 mM GC Stock+5 mL SR097 (w/o Dex); and iv)0.1 μM GC Medium: 0.5 μL of 1 mM GC Stock+5 mL SR097 (w/o Dex). A 40 μMdexamethasone control sample was run in comparison to these variousconcentrations of the alternative glucocorticoids. Additional controlsincluded Growth Medium containing 1 μl/ml dimethylsulfoxide (DMSO) as asolvent control and Growth Medium without any glucocorticoids.

Each assay was performed by using two (2) microwell plates for eachglucocorticoid tested. The sample layouts for each plate are identifiedbelow:

A. Plate One

A B C D E F G H I J K L M N O A Positive (GM containing dexamethasone) BReference (GM containing dexamethasone) C Normal (GM containingdexamethasone) D Positive (GM containing 100 μM glucocorticoid) EReference (GM containing 100 μM glucocorticoid) F Normal (GM containing100 μM glucocorticoid) G Positive (GM containing 50 μM glucocorticoid) HReference (GM containing 50 μM glucocorticoid) I Normal (GM containing50 μM glucocorticoid) J Positive (GM containing 10 μM glucocorticoid) KReference (GM containing 10 μM glucocorticoid) L Normal (GM containing10 μM glucocorticoid) M Positive (GM containing 1 μM glucocorticoid) NNormal (GM containing 1 μM glucocorticoid) O Reference (GM containing 1μM glucocorticoid)

B. Plate Two

P Q R S T U V W X Y Z AA P Positive (GM containing dexamethasone) QReference (GM containing dexamethasone) R Normal (GM containingdexamethasone) S Positive (GM containing 0.1 μM glucocorticoid) TReference (GM containing 0.1 μM glucocorticoid) U Normal (GM containing0.1 μM glucocorticoid) V Positive (GM containing DMSO) W Reference (GMcontaining DMSO) X Normal (GM containing DMSO) Y Positive (GM only) ZReference (GM only) AA Normal (GM only)

The raw data is presented below. See Table 8.

TABLE 8 Alternative Glucocortiocids - Raw Data Δ Δ (positive - normal)(positive - normal) Condition RLU SRR % s Fluticasone Proprionate 0.1 μM9768 161 1 μM 8806 148 10 μM 8774 168 50 μM 6996 137 100 μM 6366 155Prednisone 0.1 μM 8717 150 1 μM 8219 133 10 μM 8429 142 50 μM 8107 165100 μM Not Determined Not Determined Hydrocortisone 0.1 μM 9889 158 1 μM9149 155 10 μM 9757 153 50 μM 8950 178 100 μM 8348 180 Cortisone 0.1 μM10494  169 1 μM 9474 151 10 μM 9847 151 50 μM 8738 162 100 μM 8106 176Controls 40 μM Dexamethasone 9375 282 GM + 1 μL/ml DMSO 9467 141 GM w/oDexamethasone 9010 157

We claim:
 1. A method, comprising: a) providing; i) a cell line comprising a stably transfected recombinant plasmid vector encoding a chimeric TSH receptor and a reporter gene, wherein said chimeric TSH receptor is encoded by a nucleic acid sequence comprising SEQ ID NO: 3; ii) a cell culture medium compatible with said cell line; and iii) a serum sample derived from a patient suspected of having Graves' disease; b) contacting the serum sample with the cell line and the medium under conditions such that a reporter gene emits a detectable signal upon induction by a TSH receptor-specific stimulating auto-antibody.
 2. The method of claim 1, wherein an intensity of said detectable signal correlates with clinical activity.
 3. The method of claim 1, wherein an intensity of said detectable signal correlates with the clinical severity of Graves' disease.
 4. The method of claim 3 wherein said clinical severity is assessed on the basis of diplopia, proptosis, visual acuity, ocular motility, optic neuropathy, or extra-ocular muscle thickness.
 5. The method of claim 3, wherein said clinical severity is measured on the basis of the NOSPECS score.
 6. The method of claim 1, wherein an intensity of said detectable signal correlates with Graves' orbitopathy.
 7. The method of claim 1, wherein the thyrotropin stimulating hormone receptor autoantibody concentration correlates with clinical activity.
 8. The method of claim 1, wherein the thyrotropin stimulating hormone receptor autoantibody concentration correlates with the clinical severity of Graves' disease.
 9. The method of claim 8, wherein the clinical severity is assessed on the basis of diplopia, proptosis, visual acuity, ocular motility, optic neuropathy, or extra-ocular muscle thickness.
 10. The method of claim 8, wherein said clinical severity is measured on the basis of the NOSPECS score.
 11. The method of claim 1, wherein the thyrotropin stimulating hormone receptor autoantibody concentration correlates with Graves' orbitopathy.
 12. The method of claim 1, wherein said culture medium contains a glucocorticoid.
 13. The method of claim 1, wherein said reporter gene is luciferase.
 14. A method, comprising: a) providing: i) a cell line comprising a stably transfected recombinant plasmid vector encoding a chimeric TSH receptor and a reporter gene, wherein said chimeric TSH receptor is encoded by a nucleic acid sequence comprising SEQ ID NO: 3; ii) a cell culture medium compatible with said cell line; and iii) an undiluted serum sample and a diluted serum sample from a patient suspected of having Graves' disease; b) contacting the undiluted serum sample and diluted serum sample with the cell line and the medium under conditions such that a reporter gene emits a detectable signal upon induction by a TSH receptor-specific stimulating auto-antibody.
 15. The method of claim 14, wherein said method further comprises step c) comparing an intensity of said detectable signal from said undiluted sample with said diluted sample.
 16. The method of claim 15, wherein said activity from undiluted and diluted serum demonstrates a comparative reactivity profile indicative of clinical activity.
 17. The method of claim 15, wherein said activity from undiluted and diluted serum demonstrates a comparative reactivity profile indicative of severity of Graves' disease.
 18. The method of claim 15, wherein said activity from undiluted and diluted serum demonstrates a comparative reactivity profile indicative for Graves' orbitopathy.
 19. The method of claim 14, wherein said culture medium contains a glucocorticoid.
 20. The method of claim 14, wherein said reporter gene is luciferase.
 21. A method, comprising: a) providing; i) a cell line comprising a stably transfected recombinant plasmid vector encoding a chimeric TSH receptor and a reporter gene, wherein said chimeric TSH receptor is encoded by a nucleic acid sequence comprising SEQ ID NO: 3; ii) a cell culture medium compatible with said cell line; iii) a first undiluted serum sample obtained from a patient having Graves' disease prior to being treated with drug therapy; and iv) a second undiluted serum sample obtained from a patient having Graves' disease after being treated with drug therapy; b) diluting a portion of said first serum to create a diluted first serum sample; and c) contacting said undiluted first serum sample and said diluted first serum sample with the cell line and the medium under conditions such that a reporter gene emits a detectable signal upon induction by a TSH receptor-specific stimulating auto-antibody.
 22. The method of claim 21, wherein said method further comprises step d) diluting a portion of said second serum sample to create a diluted second serum sample; and e) contacting the undiluted second serum sample and diluted second serum sample with the cell line and the medium under conditions such that a reporter gene emits a detectable signal upon induction by a TSH receptor-specific stimulating auto-antibody.
 23. The method of claim 22, further comprising step f) comparing the activity of the expressed reporter gene from the first and second serum samples.
 24. The method of claim 23, wherein the comparative reactivity profiles of the first and second serum samples serve as an indication of patient response to drug treatment.
 25. The method of claim 21, wherein said culture medium contains a glucocorticoid.
 26. The method of claim 21, wherein said reporter gene is luciferase.
 27. The method of claim 21, wherein the drug received by said patients is selected from the group consisting of anti-thyroid drugs, steroids, T4, and immunosuppressive drugs.
 28. The method of claim 21, wherein the thyrotropin stimulating hormone receptor auto-antibody concentration is used to monitor drug treatment. 